Nucleic acid molecules encoding transmembrane serine proteases, the encoded proteins and methods based thereon

ABSTRACT

Provided herein is are polypeptides that include the protease domain of a type II transmembrane serine protease (MTSP) as a single chain. Methods using the polypeptides to identify compounds that modulate the protease activity of an MTSP are provided. Also provided are MTSPs designated MTSP3 and MTSP4 and a form of an MTSP designated MTSP6.

RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No.09/776,191, now allowed, to Edwin L. Madison, Edgar O. Ong andJiunn-Chern Yeh, filed on Feb. 2, 2001, entitled “NUCLEIC ACID MOLECULESENCODING TRANSMEMBRANE SERINE PROTEASES, THE ENCODED PROTEINS ANDMETHODS BASED THEREON,” which claims the benefit of priority under 35U.S.C. §119(e) to U.S. provisional application Ser. No. 60/179,982, toEdwin L. Madison and Edgar O. Ong, filed Feb. 3, 2000, entitled“NUCLEOTIDE AND PROTEIN SEQUENCES OF A TRANSMEMBRANE SERINE PROTEASE ANDMETHODS BASED THEREOF”; to U.S. provisional application Ser. No.60/183,542, to Edwin L. Madison and Edgar O. Ong, filed Feb. 18, 2000,entitled “NUCLEOTIDE AND PROTEIN SEQUENCES OF A TRANSMEMBRANE SERINEPROTEASE AND METHODS BASED THEREOF”; to U.S. provisional applicationSer. No. 60/213,124, to Edwin L. Madison and Edgar O. Ong, filed Jun.22, 2000, entitled “NUCLEOTIDE AND PROTEIN SEQUENCES OF A TRANSMEMBRANESERINE PROTEASE AND METHODS BASED THEREOF”; to U.S. provisionalapplication Ser. No. 60/220,970, to Edwin L. Madison and Edgar O. Ong,filed Jul. 26, 2000, entitled “NUCLEOTIDE AND PROTEIN SEQUENCES OF ATRANSMEMBRANE SERINE PROTEASE AND METHODS BASED THEREOF”; and to U.S.provisional application Ser. No. 60/234,840 to Edwin L. Madison, EdgarO. Ong and Jiunn-Chern Yeh, filed Sep. 22, 2000, entitled “NUCLEIC ACIDMOLECULES ENCODING TRANSMEMBRANE SERINE PROTEASES, THE ENCODED PROTEINSAND METHODS BASED THEREON” is claimed herein. Benefit of priority under35 U.S.C. §120 to U.S. application Ser. No. 09/657,986, to Edwin L.Madison, Joseph Edward Semple, Gary Samuel Coombs, John Eugene Reiner,Edgar O. Ong, Gian Luca Araldi, filed Sep. 8, 2000, entitled “INHIBITORSOF SERINE PROTEASE ACTIVITY OF MATRIPTASE OR MTSP1,” now U.S. Pat. No.6,797,504, is also claimed herein. For international purposes, benefitof priority to each of the above-noted applications is claimed herein.

This application is related to U.S. provisional application Ser. No.60/166,391 to Edwin L. Madison and Edgar O. Ong, filed Nov. 18, 1999entitled “NUCLEOTIDE AND PROTEIN SEQUENCES OF PROTEASE DOMAINS OFENDOTHELIASE AND METHODS BASED THEREON”. This application is alsorelated to International PCT application No. PCT/US00/31803, filed Nov.17, 2000.

The above-noted provisional applications, patent applications andInternational PCT application are incorporated by reference in theirentirety. All patents, applications, published applications and otherpublications and sequences from GenBank and other data bases referred toherein are incorporated by reference in their entirety.

FIELD OF INVENTION

Nucleic acid molecules that encode proteases and portions thereof,particularly protease domains are provided. Also provided areprognostic, diagnostic and therapeutic methods using the proteases anddomains thereof and the encoding nucleic acid molecules.

BACKGROUND OF THE INVENTION AND OBJECTS THEREOF

Cancer a leading cause of death in the United States, developing in onein three Americans; one of every four Americans dies of cancer. Canceris characterized by an increase in the number of abnormal neoplasticcells, which proliferate to form a tumor mass, the invasion of adjacenttissues by these neoplastic tumor cells, and the generation of malignantcells that metastasize via the blood or lymphatic system to regionallymph nodes and to distant sites.

Among the hallmarks of cancer is a breakdown in the communication amongtumor cells and their environment. Normal cells do not divide in theabsence of stimulatory signals, cease dividing in the presence ofinhibitory signals. Growth-stimulatory and growth-inhibitory signals,are routinely exchanged between cells within a tissue. In a cancerous,or neoplastic, state, a cell acquires the ability to “override” thesesignals and to proliferate under conditions in which normal cells do notgrow.

In order to proliferate tumor cells acquire a number of distinctaberrant traits reflecting genetic alterations. The genomes of certainwell-studied tumors carry several different independently altered genes,including activated oncogenes and inactivated tumor suppressor genes.Each of these genetic changes appears to be responsible for impartingsome of the traits that, in the aggregate, represent the full neoplasticphenotype.

A variety of biochemical factors have been associated with differentphases of metastasis. Cell surface receptors for collagen, glycoproteinssuch as laminin, and proteoglycans, facilitate tumor cell attachment, animportant step in invasion and metastases. Attachment triggers therelease of degradative enzymes which facilitate the penetration of tumorcells through tissue barriers. Once the tumor cells have entered thetarget tissue, specific growth factors are required for furtherproliferation. Tumor invasion (or progression) involves a complex seriesof events, in which tumor cells detach from the primary tumor, breakdown the normal tissue surrounding it, and migrate into a blood orlymphatic vessel to be carried to a distant site. The breaking down ofnormal tissue barriers is accomplished by the elaboration of specificenzymes that degrade the proteins of the extracellular matrix that makeup basement membranes and stromal components of tissues.

A class of extracellular matrix degrading enzymes have been implicatedin tumor invasion. Among these are the matrix metalloproteinases (MMP).For example, the production of the matrix metalloproteinase stromelysinis associated with malignant tumors with metastatic potential (see,e.g., Matrisian et al. (1990) Smnrs. in Cancer Biology 1:107-115;McDonnell et al. (1990) Cancer and Metastasis Reviews 9:309-319).

The capacity of cancer cells to metastasize and invade tissue isfacilitated by degradation of the basement membrane. Several proteinaseenzymes, including the MMPs, have been reported to facilitate theprocess of invasion of tumor cells. MMPs are reported to enhancedegradation of the basement membrane, which thereby permits tumorouscells to invade tissues. For example, two major metalloproteinaseshaving molecular weights of about 70 kDa and 92 kDa appear to enhanceability of tumor cells to metastasize.

Type II Transmembrane Serine Proteases (TTSPs)

In addition to the MMPs, serine proteases have been implicated inneoplastic disease progression. Most serine proteases, which are eithersecreted enzymes or are sequestered in cytoplasmic storage organelles,have roles in blood coagulation, wound healing, digestion, immuneresponses and tumor invasion and metastasis. A class cell surfaceproteins designated type II transmembrane serine proteases, which aremembrane-anchored proteins with N-terminal extracellular domains, hasbeen identified. As cell surface proteins, they are positioned to play arole in intracellular signal transduction and in mediating cell surfaceproteolytic events.

Cell surface proteolysis is a mechanism for the generation ofbiologically active proteins that mediate a variety of cellularfunctions. These membrane-anchored proteins, include a disintegrin-likeand metalloproteinase (ADAM) and membrane-type matrix metalloproteinase(MT-MMP). In mammals, at least 17 members of the family are known,including seven in humans (see, Hooper et al. (2001) J. Biol. Chem.276:857-860). These include: corin (accession nos. AF133845 andAB013874; see, Yan et al. (1999) J. Biol. Chem. 274:14926-14938; Tomiaet at (1998) J. Biochem. 124:784-789; Yan et at (2000) Proc. Natl. Acad.Sci. U.S.A. 97:8525-8529); enterpeptidase (also designated enterokinase;accession no. U09860 for the human protein; see, Kitamoto et al. (1995)Biochem. 27:4562-4568; Yahagi et al. (1996) Biochem. Biophys. Res.Commun. 219:806-812; Kitamoto et al. (1994) Proc. Natl. Acad. Sci.U.S.A. 91:7588-7592; Matsushima et at (1994) J. Biol. Chem.269:19976-19982;); human airway trypsin-like protease (HAT; accessionno. A80021 34; see Yamaoka et al. J. Biol. Chem. 273:11894-11901); MTSP1and matriptase (also called TADG-15; see SEQ ID Nos. 1 and 2; accessionnos. AFI 33086/AF118224, AF04280022; Takeuchi et at (1999) Proc. Natl.Acad. Sci. U.S.A. 96:11054-1161; Lin et al. (1999) J. Biol. Chem.274:18231-18236; Takeuchi et at (2000) J. Biol. Chem. 275:26333-26342;and Kim et al. (1999) Immunogenetics 49:420-429); hepsin (see, accessionnos. M18930, AF030065, X70900; Leytus et al. (1988) Biochem.27:11895-11901; Vu et al. (1997) J. Biol. Chem. 272:31315-31320; andFarley et al. (1993) Biochem. Biophys. Acta 1173:350-352; and see, U.S.Pat. No. 5,972,616); TMPRS2 (see, Accession Nos. U75329 and AF113596;Paoloni-Giacobino et al. (1997) Genomics 44:309-320; and Jacquinet etal. (2000) FEBS Lett. 468:93-100); and TMPRSS4 (see, Accession No. NM016425; Wallrapp et al. (2000) Cancer 60: 2602-2606).

Serine proteases, including transmembrane serine proteases, have beenimplicated in processes involved in neoplastic development andprogression. While the precise role of these proteases has not beenelaborated, serine proteases and inhibitors thereof are involved in thecontrol of many intra- and extracellular physiological processes,including degradative actions in cancer cell invasion, metastaticspread, and neovascularization of tumors, that are involved in tumorprogression. It is believed that proteases are involved in thedegradation of extracellular matrix (ECM) and contribute to tissueremodeling, and are necessary for cancer invasion and metastasis. Theactivity and/or expression of some proteases have been shown tocorrelate with tumor progression and development.

For example, a membrane-type serine protease MTSP1 (also calledmatriptase; see SEQ ID Nos. 1 and 2 from U.S. Pat. No. 5,972,616; andGenBank Accession No. AF118224; (1999) J. Biol. Chem. 274:18231-18236;U.S. Pat. No. 5,792,616; see, also Takeuchi (1999) Proc. Natl. Acad.Sci. U.S.A. 96:11054-1161) that is expressed in epithelial cancer andnormal tissue (Takeucuhi et al. (1999) Proc. Natl. Acad. Sci. USA,96(20):11054-61) has been identified. Matriptase was originallyidentified in human breast cancer cells as a major gelatinase (see, U.S.Pat. No. 5,482,848), a type of matrix metalloprotease (MMP). It has beenproposed that it plays a role in the metastasis of breast cancer. Itsprimary cleavage specificity is Arg-Lys residues. Matriptase also isexpressed in a variety of epithelial tissues with high levels ofactivity and/or expression in the human gastrointestinal tract and theprostate.

Prostate-specific antigen (PSA), a kallikrein-like serine protease,degrades extracellular matrix glycoproteins fibronectin and laminin,and, has been postulated to facilitate invasion by prostate cancer cells(Webber et al. (1995) Clin. Cancer Res., 1(10):1089-94). Blocking PSAproteolytic activity with PSA-specific monoclonal antibodies results ina dose-dependent decrease in vitro in the invasion of the reconstitutedbasement membrane Matrigel by LNCaP human prostate carcinoma cells whichsecrete high levels of PSA.

Hepsin, a cell surface serine protease identified in hepatoma cells, isoverexpressed in ovarian cancer (Tanimoto et al. (1997) Cancer Res.,57(14):2884-7). The hepsin transcript appears to be abundant incarcinoma tissue and is almost never expressed in normal adult tissue,including normal ovary. It has been suggested that hepsin is frequentlyoverexpressed in ovarian tumors and therefore may be a candidateprotease in the invasive process and growth capacity of ovarian tumorcells.

A serine protease-like gene, designated normal epithelial cell-specific1 (NES1) (Liu et al., Cancer Res., 56(14):3371-9 (1996)) has beenidentified. Although expression of the NES1 mRNA is observed in allnormal and immortalized nontumorigenic epithelial cell lines, themajority of human breast cancer cell lines show a drastic reduction or acomplete lack of its expression. The structural similarity of NES1 topolypeptides known to regulate growth factor activity and a negativecorrelation of NES1 expression with breast oncogenesis suggest a director indirect role for this protease-like gene product in the suppressionof tumorigenesis.

Hence transmembrane serine proteases appear to be involved in theetiology and pathogenesis of tumors. There is a need to furtherelucidate their role in these processes and to identify additionaltransmembrane proteases. Therefore, it is an object herein to providetransmembrane serine protease (MTSP) proteins and nucleic acids encodingsuch MTSP proteases that are involved in the regulation of orparticipate in tumorigenesis and/or carcinogenesis. It is also an objectherein to provide prognostic, diagnostic, therapeutic screening methodsusing the such proteases and the nucleic acids encoding such proteases.

SUMMARY OF THE INVENTION

Provided herein are isolated protease domains of the TransmembraneSerine Protease family, particularly the Type II Transmembrane SerineProtease (TTSP) family (also referred to herein as MTSPs), and moreparticularly TTSP family members whose functional activity differs intumor cells from non-tumor cells in the same tissue. For example, theMTSPs include those that are activated and/or expressed in tumor cellsat different levels, typically higher, from non-tumor cells; and thosefrom cells in which substrates therefor differ in tumor cells fromnon-tumor cells or otherwise alter the specificity of the MTSP.

The MTSP family as intended herein does not include any membraneanchored or spanning proteases that are expressed on endothelial cells.Included among the MTSPs are several heretofore unidentified MTSP familymembers, designated herein as MTSP3 and MTSP4 and a new form of aprotein designated herein as MTSP6. In addition to the protease domainsof each of MTSP3 and MTSP4, the full-length proteins, including thosethat results from splice variants, zymogens and activated forms, anduses thereof, are also provided.

The protease domains as provided herein are single-chain polypeptides,with an N-terminus (such as IV, VV, IL and II) generated at the cleavagesite (generally having the consensus sequence R↓VVGG, R↓IVGG, R↓IVNG,R↓ILGG, R↓VGLL, R↓ILGG or a variation thereof; an N-terminus R↓V or R↓I,where the arrow represents the cleavage point) when the zymogen isactivated. To identify the protease domain an RI should be identified,and then the following amino acids compared to the above noted motif.

The protease domains generated herein, however, do not result fromactivation, which produces a two chain activated product, but rather aresingle chain polypeptides with the N-terminus include the consensussequence ↓VVGG, ↓IVGG, ↓VGLL, ↓ILGG or ↓IVNG or other such motif at theN-terminus. As shown herein, such polypeptides, although not the resultof activation and not double-chain forms, exhibit proteolytic.(catalytic) activity. These protease domain polypeptides are used inassays to screen for agents that modulate the activity of the MTSP. Suchassays are also provided herein. In exemplary assays, the affects oftest compounds in the ability of a protease domains to proteolyticallycleave a known substrate, typically a fluorescently, chromogenically orotherwise detectably labeled substrate, are assessed. Agents, generallycompounds, particularly small molecules, that modulate the activity ofthe protease domain are candidate compounds for modulating the activityof the MTSP. The protease domains can also be used to producesingle-chain protease-specific antibodies. The protease domains providedherein include, but are not limited to, the single chain region havingan N-terminus at the cleavage site for activation of the zymogen,through the C-terminus, or C-terminal truncated portions thereof thatexhibit proteolytic activity as a single-chain polypeptide in in vitroproteolysis assays, of any MTSP family member, preferably from a mammal,including and most preferably human, that, for example, is expressed intumor cells at different levels from non-tumor cells, and that is notexpressed on an endothelial cell. These include, but are not limited to:MTSP1 (or matriptase), MTSP3, MTSP4 and MTSP6. Other MTSP proteasedomains of interest herein, particularly for use in in vitro drugscreening proteolytic assays, include, but are not limited to: corin(accession nos. AF133845 and AB013874; see, Yan et al (1999) J. Biol.Chem. 274:14926-14938; Tomia et al. (1998) J. Biochem. 124:784-789; Yanet al. (2000) Proc. Natl. Acad. Sci. U.S.A. 97:8525-8529; SEQ ID Nos. 61and 62 for the human protein); enterpeptidase (also designatedenterokinase; accession no. U09860 for the human protein; see, Kitamotoet al. (1995) Biochem. 27:4562-4568; Yahagi et al. (1996) Biochem.Biophys. Res. Commun. 219:806-812; Kitamoto et al. (1994) Proc. Natl.Acad. Sci. U.S.A. 91:7588-7592; Matsushima et al. (1994) J. Biol. Chem.269:19976-19982; see SEQ ID Nos. 63 and 64 for the human protein); humanairway trypsin-like protease (HAT; accession no. AB002134; see Yamaokaet al. J. Biol. Chem. 273:11894-11901; SEQ ID Nos. 65 and 66 for thehuman protein); hepsin (see, accession nos. M18930, AF030065, X70900;Leytus et al. (1988) Biochem. 27:11895-11901; Vu et al. (1997) J. Biol.Chem. 272:31315-31320; and Farley et al. (1993) Biochem. Biophys. Acta1173:350-352; SEQ ID Nos. 67 and 68 for the human protein); TMPRS2 (see,Accession Nos. U75329 and AF113596; Paoloni-Giacobino et al. (1997)Genomics 44:309-320; and Jacquinet et al. (2000) FEBS Lett. 468:93-100;SEQ ID Nos. 69 and 70 for the human protein) TMPRSS4 (see, Accession No.NM 016425; Wallrapp et al. (2000) Cancer 60:2602-2606; SEQ ID Nos. 71and 72 for the human protein); and TADG-12 (also designated MTSP6, seeSEQ ID Nos. 11 and 12; see International PCT application No. WO00/52044, which claims priority to U.S. application Ser. No.09/261,416).

Also provided are muteins of the single chain protease domains andMTSPs, particularly muteins in which the Cys residue in the proteasedomain that is free (i.e., does not form disulfide linkages with anyother Cys residue in the protein) is substituted with another amino acidsubstitution, preferably with a conservative amino acid substitution ora substitution that does not eliminate the activity, and muteins inwhich a glycosylation site(s) is eliminated. Muteins in which otherconservative amino acid substitutions in which catalytic activity isretained are also contemplated (see, e.g., Table 1, for exemplary aminoacid substitutions). See, also, FIG. 4, which identifies the free Cysresidues in MTSP3, MTSP4 and MTSP6.

Hence, provided herein are members of a family of transmembrane serineprotease (MTSP) proteins, and functional domains, especially protease(or catalytic) domains thereof, muteins and other derivatives andanalogs thereof. Also provided herein are nucleic acids encoding theMTSPs.

Exemplary MTSPs (see, e.g., SEQ ID No. 1-12, 49 and 50) are providedherein, as are the single chain protease domains thereof as follows: SEQID Nos. 1, 2, 49 and 50 set forth amino acid and nucleic acid sequencesof MTSP1 and the protease domain thereof; SEQ ID No. 3 sets forth theMTSP3 nucleic acid sequence and SEQ ID No. 4 the encoded MTSP3 aminoacids; SEQ ID No. 5 MTSP4 a nucleic acid sequence of the protease domainand SEQ ID No. 6 the encoded MTSP4 amino acid protease domain; SEQ IDNo. 7 MTSP4-L a nucleic acid sequence and SEQ ID No. 8 the encodedMTSP4-L amino acid sequence; SEQ ID No. 9 an MTSP4-S encoding nucleicacid sequence and SEQ ID No. 10 the encoded MTSP4-S amino acid sequence;and SEQ ID No. 11 an MTSP6 encoding nucleic acid sequence and SEQ ID No.12 the encoded MTSP6 amino acid sequence. The single chain proteasedomains of each are delineated below.

Nucleic acid molecules that encode a single-chain protease domain orcatalytically active portion thereof are provided. Also provided arenucleic acid molecules that hybridize to such MTSP encoding nucleic acidalong their full length and encode the protease domain or portionthereof are provided. Hybridization is preferably effected underconditions of at least low, generally at least moderate, and often highstringency.

Additionally provided herein are antibodies that specifically bind tothe MTSPs, cells, combinations, kits and articles of manufacture thatcontain the nucleic acid encoding the MTSP and/or the MTSP. Furtherprovided herein are prognostic, diagnostic, therapeutic screeningmethods using MTSPs and the nucleic acids encoding MTSP. Also providedare transgenic non-human animals bearing inactivated genes encoding theMTSP and bearing the genes encoding the MTSP under non-native promotorcontrol. Such animals are useful in animal models of tumor initiation,growth and/or progression models.

Provided herein are members of a family of membrane serine proteases(MTSP) that are expressed in certain tumor or cancer cells such lung,prostate, colon and breast cancers. In particular, it is shown herein,that MTSPs, particularly, MTSP3, MTSP4 and MTSP6 are expressed in lungcarcinoma, breast carcinoma, colon adenocarcinoma and/or ovariancarcinomas as well as in certain normal cells and tissues (see e.g.,EXAMPLES for tissue-specific expression profiles of each proteinexemplified herein). The MTSPs that are of particular interest herein,are those that are expressed in tumor cells, for example, those thatappear to be expressed at different levels in tumor cells from normalcells, or whose functional activity is different in tumor cells fromnormal cells, such as by an alteration in a substrate therefor, or acofactor. Hence the MTSP provided herein can serve as diagnostic markersfor certain tumors. The level of activated MTSP3, MTSP4 and MTSP6 can bediagnostic of prostate cancer. In addition, MTSP4 is expressed and/oractivated in lymphomas, leukemias, lung cancer, breast, prostrate andcolon cancers. MTSP6 is activated and/or expressed in breast, lung,prostate, colon and ovarian cancers. Furthermore, compounds thatmodulate the activity of these MTSPs, as assessed by the assays providedherein, particularly the in vitro proteolytic assays that use the singlechain protease domains, are potential therapeutic candidates fortreatment of various malignancies and neoplastic disease.

Also provided herein are methods of modulating the activity of the MTSPsand screening for compounds that modulate, including inhibit,antagonize, agonize or otherwise alter the activity of the MTSPs. Ofparticular interest is the extracellular domain of these MTSPs thatincludes the proteolytic (catalytic) portion of the protein.

MTSP proteins, including, but not limited to, MTSP3, MTSP4, and MTSP6,including splice variants thereof, and nucleic acids encoding MTSPs, anddomains, derivatives and analogs thereof are provided herein. Singlechain protease domains, in the N-terminal is that which would begenerated by activation of the zymogen, from any MTSP, particularlythose that are not expressed in endothelial cells and that are expressedin tumor cells are also provided.

Antibodies that specifically bind to the MTSP, particularly the singlechain protease domain, and any and all forms of MTSP3 and MTSP4, andcells, combinations, kits and articles of manufacture containing theMTSP proteins, domains thereof, or encoding nucleic acids are alsoprovided herein. Transgenic non-human animals bearing inactivated genesencoding the MTSP and bearing the genes encoding the MTSP under anon-native promotor control are additionally provided herein. Alsoprovided are nucleic acid molecules encoding each of the MTSPs anddomains thereof.

Also provided are plasmids containing any of the nucleic acid moleculesprovided herein. Cells containing the plasmids are also provided. Suchcells include, but are not limited to, bacterial cells, yeast cells,fungal cells, plant cells, insect cells and animal cells.

Also provided is a method of producing a MTSP by growing theabove-described cells under conditions whereby the MTSP is expressed bythe cells, and recovering the expressed MTSP protein. Methods forisolating nucleic acid encoding other MTSPs are also provided.

Also provided are cells, preferably eukaryotic cells, such as mammaliancells and yeast cells, in which the MTSP protein, preferably MTSP3 andMTSP4, is expressed in the surface of the cells. Such cells are used indrug screening assays to identify compounds that modulate the activityof the MTSP protein. These assays including in vitro binding assays, andtranscription based assays in which signal transduction mediated by theMTSP is assessed.

Further provided herein are prognostic, diagnostic and therapeuticscreening methods using the MTSP and the nucleic acids encoding MTSP. Inparticular, the prognostic, diagnostic and therapeutic screening methodsare used for preventing, treating, or for finding agents useful inpreventing or treating, tumors or cancers such as lung carcinoma, colonadenocarcinoma and ovarian carcinoma.

Also provided are methods for screening for compounds that modulate theactivity of any MTSP. The compounds are identified by contacting themwith the MTSP and a substrate for the MTSP. A change in the amount ofsubstrate cleaved in the presence of the compounds compared to that inthe absence of the compound indicates that the compound modulates theactivity of the MTSP. Such compounds are selected for further analysesor for use to modulate the activity of the MTSP, such as inhibitors oragonists. The compounds can also be identified by contacting thesubstrates with a cell that expresses the MTSP or the extracellulardomain or proteolytically active portion thereof. For assays in whichthe extracellular domain or a proteolytically active portion thereof isemployed, the MTSP is any MTSP that is expressed on cells, other thanendothelial cells, including, but not limited to MTSP1, MTSP3, MTSP4 andMTSP6.

Also provided herein are modulators of the activity of the MTSP,especially the modulators obtained according to the-screening methodsprovide herein. Such modulators may have use in treating cancerousconditions, and other neoplastic conditions.

Pharmaceutical composition containing the protease domains of an MTSPprotein, and the MTSP proteins, MTSP3, MTSP4 and MTSP6 are providedherein in a pharmaceutically acceptable carrier or excipient areprovided herein.

Also provided are articles of manufacture that contain the MTSP proteinsand protease domains of MTSPs in single chain form. The articles containa) packaging material; b) the polypeptide (or encoding nucleic acid),particularly the single chain protease domain thereof; and c) a labelindicating that the article is for using ins assays for identifyingmodulators of the activities of an MTSP protein is provided herein.

Conjugates containing a) a MTSP protease domain in single chain from;and b) a targeting agent linked to the MTSP directly or via a linker,wherein the agent facilitates: i) affinity isolation or purification ofthe conjugate; ii) attachment of the conjugate to a surface; iii)detection of the conjugate; or iv) targeted delivery to a selectedtissue or cell, is provided herein. The conjugate can contain aplurality of agents linked thereto. The conjugate can be a chemicalconjugate; and it can be a fusion protein.

In yet another embodiment, the targeting agent is a protein or peptidefragment. The protein or peptide fragment can include a protein bindingsequence, a nucleic acid binding sequence, a lipid binding sequence, apolysaccharide binding sequence, or a metal binding sequence.

Method of diagnosing a disease or disorder characterized by detecting anaberrant level of an MTSP, particularly an MTSP3, MTSP4 or MTSP6, in asubject is provided. The method can be practiced by measuring the levelof the DNA, RNA, protein or functional activity of the MTSP. An increaseor decrease in the level of the DNA, RNA, protein or functional activityof the MTSP, relative to the level of the DNA, RNA, protein orfunctional activity found in an analogous sample not having the diseaseor disorder (or other suitable control) is indicative of the presence ofthe disease or disorder in the subject or other relative any othersuitable control.

Combinations are provided herein. The combination can include: a) aninhibitor of the activity of an MTSP; and b) an anti-cancer treatment oragent. The MTSP inhibitor and the anti-cancer agent can be formulated ina single pharmaceutical composition or each is formulated in a separatepharmaceutical composition. The MTSP inhibitor can be an antibody or afragment or binding portion thereof against the MTSP, such as anantibody that specifically binds to the protease domain, an inhibitor ofthe MTSP production, or an inhibitor of the MTSP membrane-localizationor an inhibitor of MTSP activation. Other MTSP inhibitors include, butare not limited to, an antisense nucleic acid encoding the MTSP,particularly a portion of the protease domain; a nucleic acid encodingat least a portion of a gene encoding the MTSP with a heterologousnucleotide sequence inserted therein such that the heterologous sequenceinactivates the biological activity encoded MTSP or the gene encodingit. The portion of the gene encoding the MTSP preferably flanks theheterologous sequence to promote homologous recombination with a genomicgene encoding the MTSP.

Also, provided are methods for treating or preventing a tumor or cancerin a mammal by administering to a mammal an effective amount of aninhibitor of an MTSP3, MTSP4 or MTSP6, whereby the tumor or cancer istreated or prevented. The MTSP inhibitor used in the treatment or forprophylaxis is administered with a pharmaceutically acceptable carrieror excipient. The mammal treated can be a human. The treatment orprevention method can additionally include administering an anti-cancertreatment or agent simultaneously with or subsequently or beforeadministration of the MTSP inhibitor.

Also provided is a recombinant non-human animal in which an endogenousgene of an MTSP has been deleted or inactivated by homologousrecombination or insertional mutagenesis of the animal or an ancestorthereof. A recombinant non-human animal is provided herein, where thegene of an MTSP is under control of a promoter that is not the nativepromoter of the gene or that is not the native promoter of the gene inthe non-human animal or where the nucleic acid encoding the MTSP isheterologous to the non-human animal and the promoter is the native or anon-native promoter.

Also provided are methods of treatments of tumors by administering aprodrug that is activated by an MTSP that is expressed or active intumor cells, particularly those in which its functional activity intumor cells is greater than in none-tumor cells. The prodrug isadministered and, upon administration, active MTSP expressed on cellscleaves the prodrug and releases active drug in the vicinity of thesecells. The active anti-cancer drug accumulates in the vicinity of thetumor. This is particularly useful in instances in which an MTSP isexpressed or active in greater quantity, higher level or predominantlyin tumor cells compared to other cells.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 illustrates the domain organization of the MTSP3;

FIG. 2 illustrates the domain organization of the MTSP4 splice variantsand domains thereof; MTSP4-L includes a transmembrane domain, a CUBdomain, a low density lipoprotein receptor (LDLR) domains, and a serineprotease catalytic domain; MTSP4-S lacking the portion between aminoacids 136-279.

FIG. 3 depicts the domain organization of MTSP6.

FIG. 4 provides an alignment of the C-terminal portions of MTSP3 (setforth herein as SEQ ID No. 4), the two splice variant-encoded forms ofMTSP4 (MTSP4-L and MTSP4-S set forth herein as SEQ ID Nos. 8 and 10,respectively), and MTSP6 (set forth herein as SEQ ID No. 12), thatencompasses the protease domains thereof; the figure shows the cleavagesites, which form the N-terminus of the protease domain of each protein;a potential glycosylation site is noted and the free Cys residues in theprotease domain of each are noted (*). Muteins of each protein may beprepared by replacing the residues in the glycosylation site,particularly the N residue, and the free Cys residues, with preferablyconservative amino acid residues. Such muteins are also provided herein.

DETAILED DESCRIPTION OF THE INVENTION A. DEFINITIONS

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as is commonly understood by one of ordinary skillin the art to which this invention belongs. All patents, applications,published applications and other publications and sequences from GenBankand other data bases referred to herein are incorporated by reference intheir entirety.

As used herein, the abbreviations for any protective groups, amino acidsand other compounds, are, unless indicated otherwise, in accord withtheir common usage, recognized abbreviations, or the IUPAC-IUBCommission on Biochemical Nomenclature (see, (1972) Biochem.11:942-944).

As used herein, serine protease refers to a diverse family of proteaseswherein a serine residue is involved in the hydrolysis of proteins orpeptides. The serine residue can be part of the catalytic triadmechanism, which includes a serine, a histidine and an aspartic acid inthe catalysis, or be part of the hydroxyl/ε-amine or hydroxyl/α-aminecatalytic dyad mechanism, which involves a serine and a lysine in thecatalysis.

As used herein, “transmembrane serine protease (MTSP)” refers to afamily of transmembrane serine proteases that share common structuralfeatures as described herein (see, also Hooper et al. (2001) J. Biol.Chem. 276:857-860). Thus, reference, for example, to “MTSP” encompassesall proteins encoded by the MTSP gene family, including but are notlimited to: MTSP1, MTSP3, MTSP4 and MTSP6, or an equivalent moleculeobtained from any other source or that has been prepared syntheticallyor that exhibits the same activity. Other MTSPs include, but are notlimited to, corin, enterpeptidase, human airway trypsin-like protease(HAT), MTSP1, TMPRSS2, and TMPRSS4. Sequences of encoding nucleicmolecules and the encoded amino acid sequences of exemplary MTSPs and/ordomains thereof are set forth in SEQ ID Nos. 1-12, 49, 50 and 61-72. Theterm also encompass MTSPs with conservative amino acid substitutionsthat do not substantially alter activity of each member, and alsoencompasses splice variants thereof. Suitable conservative substitutionsof amino acids are known to those of skill in this art and may be madegenerally without altering the biological activity of the resultingmolecule. Of particular interest are MTSPs of mammalian, includinghuman, origin. Those of skill in this art recognize that, in general,single amino acid substitutions in non-essential regions of apolypeptide do not substantially alter biological activity (see, e.g.,Watson et al. Molecular Biology of the Gene, 4th Edition, 1987, TheBejacmin/Cummings Pub. co., p. 224).

As used herein, a “protease domain of an MTSP” refers to the proteasedomain of MTSP that is located within the extracellular domain of a MTSPand exhibits serine proteolytic activity. It includes at least thesmallest fragment thereof that acts catalytically as a single chainform. Hence it is at least the minimal portion of the extracellulardomain that exhibits proteolytic activity as assessed by standard assaysin vitro assays. Those of skill in this art recognize that such proteasedomain is the portion of the protease that is structurally equivalent tothe trypsin or chymotrypsin fold.

Exemplary MTSP proteins, with the protease domains indicated, areillustrated in FIGS. 1-3, Smaller portions thereof that retain proteaseactivity are contemplated. The protease domains vary in size andconstitution, including insertions and deletions in surface loops. Theyretain conserved structure, including at least one of the active sitetriad, primary specificity pocket, oxyanion hole and/or other featuresof serine protease domains of proteases. Thus, for purposes herein, theprotease domain is a portion of a MTSP, as defined herein, and ishomologous to a domain of other MTSPs, such as corin, enterpeptidase,human airway trypsin-like protease (HAT), MTSP1, TMPRSS2, and TMPRSS4,which have been previously identified; it was not recognized, however,that an isolated single chain form of the protease domain could functionproteolytically in in vitro assays. As with the larger class of enzymesof the chymotrypsin (S1) fold (see, e.g., Internet accessible MEROPSdata base), the MTSPs protease domains share a high degree of amino acidsequence identity. The His, Asp and Ser residues necessary for activityare present in conserved motifs. The activation site, which results inthe N-terminus of second chain in the two chain forms is has a conservedmotif and readily can be identified (see, e.g., amino acids 801-806, SEQID No. 62, amino acids 406-410, SEQ ID No. 64; amino acids 186-190, SEQID No. 66; amino acids 161-166, SEQ ID No. 68; amino acids 255-259, SEQID No. 70; amino acids 190-194, SEQ ID No. 72).

As used herein, the catalytically active domain of an MTSP refers to theprotease domain. Reference to the protease domain of an MTSP refersincludes the single and double-chain forms of any of these proteins. Thezymogen form of each protein is single chain form, which can beconverted to the active two chain form by cleavage. The protease domainmay also be converted to a two chain form. By active form is meant aform active in vivo.

Significantly, it is shown herein, that, at least in vitro, the singlechain forms of the MTSPs and the catalytic domains or proteolyticallyactive portions thereof (typically C-terminal truncation) thereofexhibit protease activity. Hence provided herein are isolated singlechain forms of the protease domains of MTSPs and their use in in vitrodrug screening assays for identification of agents that modulate theactivity thereof.

As used herein an MTSP3, whenever referenced herein, includes at leastone or all of or any combination of:

-   -   a polypeptide encoded by the sequence of nucleotides set forth        in SEQ ID No. 3;    -   a polypeptide encoded by a sequence of nucleotides that        hybridizes under conditions of low, moderate or high stringency        to the sequence of nucleotides set forth in SEQ ID No. 3;    -   a polypeptide that comprises the sequence of amino acids set        forth as amino acids 205-437 of SEQ ID No. 4;    -   a polypeptide that comprises a sequence of amino acids having at        least about 85% or 90% sequence identity with the sequence of        amino acids set forth in SEQ ID No. 4; and/or    -   a splice variant of the MTSP3 set forth in SEQ ID Nos. 3 and 4.

The MTSP3 may be from any animal, particularly a mammal, and includesbut are not limited to, humans, rodents, fowl, ruminants and otheranimals. The full length zymogen or double chain activated form iscontemplated or any domain thereof, including the protease domain, whichcan be a double chain activated form, or a single chain form.

As used herein an MTSP4, whenever referenced herein, includes at leastone or all of or any combination of: a polypeptide encoded by thesequence of nucleotides set forth in any of SEQ ID No. 5, 7 or 9;

-   -   a polypeptide encoded by a sequence of nucleotides that        hybridizes under conditions of low, moderate or high stringency        to the sequence of nucleotides set forth in any of SEQ ID Nos.        5, 7 or 9;    -   a polypeptide that comprises the sequence of amino acids set        forth in any of SEQ ID Nos. 6, 8 or 10;    -   a polypeptide that comprises a sequence of amino acids having at        least about 85% or 90% or 95% sequence identity with the        sequence of amino acids set forth in SEQ ID No. 6, 8 or 10;        and/or    -   a splice variant of the MTSP4s set forth in SEQ ID Nos. 7-10.

The MTSP4 may be from any animal, particularly a mammal, and includesbut are not limited to, humans, rodents, fowl, ruminants and otheranimals. The full length zymogen or double chain activated form iscontemplated or any domain thereof, including the protease domain, whichcan be a double chain activated form, or a single chain form.

As used herein an MTSP6, whenever referenced herein, includes at leastone or all of or any combination of:

-   -   a polypeptide encoded by the sequence of nucleotides set forth        in any of SEQ ID No. 11;    -   a polypeptide encoded by a sequence of nucleotides that        hybridizes under conditions of low, moderate or high stringency        to the sequence of nucleotides set forth in any of SEQ ID Nos.        11;    -   a polypeptide that comprises the sequence of amino acids set        forth in any of SEQ ID Nos. 12;    -   a polypeptide that comprises a sequence of amino acids having at        least about 90% or 95% or 98% sequence identity with the        sequence of aminoacids set forth in SEQ ID No. 12; and/or    -   a splice variant of the MTSP4s set forth in SEQ ID No. 12.

The MTSP6 may be from any animal, particularly a mammal, and includesbut are not limited to, humans, rodents, fowl, ruminants and otheranimals. The full length zymogen or double chain activated form iscontemplated or any domain thereof, including the protease domain, whichcan be a double chain activated form, or a single chain form. Ofparticular interest herein is the MTSP6 of SEQ ID No. 12.

As used herein, a human protein is one encoded by DNA present in thegenome of a human, including all allelic variants and conservativevariations as long as they are not variants found in other mammals.

As used herein, a “nucleic acid encoding a protease domain orcatalytically active portion of a MTSP” shall be construed as referringto a nucleic acid encoding only the recited single chain protease domainor active portion thereof, and not the other contiguous portions of theMTSP as a continuous sequence.

As used herein, a CUB domain is a motif that mediates protein-proteininteractions in complement components C1r/C1s and has also beenidentified in various proteins involved in developmental processes.

As used herein, catalytic activity refers to the activity of the MTSP asa serine proteases. Function of the MTSP refers to its function in tumorbiology, including promotion of or involvement in tumorigenesis,metastasis or carcinogenesis, and also roles in signal transduction.

As used herein, a “propeptide” or “pro sequence” is sequence of aminoacids positioned at the amino terminus of a mature biologically activepolypeptide. When so-positioned, the resulting polypeptide is called azymogen. Zymogens, generally, are biologically inactive and can beconverted to mature active polypeptides by catalytic or autocatalyticcleavage of the propeptide from the zymogen. A zymogen is anenzymatically inactive protein that is converted to a proteolytic enzymeby the action of an activator. Cleavage may be effectedautocatalytically.

As used herein, “disease or disorder” refers to a pathological conditionin an organism resulting from, e.g., infection or genetic defect, andcharacterized by identifiable symptoms.

As used herein, neoplasm (neoplasia) refers to abnormal new growth, andthus means the same as tumor, which may be benign or malignant. Unlikehyperplasia, neoplastic proliferation persists even in the absence ofthe original stimulus.

As used herein, neoplastic disease refers to any disorder involvingcancer, including tumor development, growth, metastasis and progression.

As used herein, cancer refers to a general term for diseases caused byany type of malignant tumor.

As used herein, malignant, as applies to tumors, refers to primarytumors that have the capacity of metastasis with loss of growth controland positional control.

As used herein, an anti-cancer agent (used interchangeable with“anti-tumor or anti-neoplastic agent”) refers to any agents used in theanti-cancer treatment. These include any agents, when used alone or incombination with other compounds, that can alleviate, reduce,ameliorate, prevent, or place or maintain in a state of remission ofclinical symptoms or diagnostic markers associated with neoplasticdisease, tumor and cancer, and can be used in methods, combinations andcompositions provided herein. Non-limiting examples of anti-neoplasticagents include anti-angiogenic agents, alkylating agents,antimetabolite, certain natural products, platinum coordinationcomplexes, anthracenediones, substituted ureas, methylhydrazinederivatives, adrenocortical suppressants, certain hormones, antagonistsand anti-cancer polysaccharides.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic DNA thatresults in more than one type of mRNA. Splice variants of MTSPs areprovided herein.

As used herein, angiogenesis is intended to broadly encompass thetotality of processes directly or indirectly involved in theestablishment and maintenance of new vasculature (neovascularization),including, but not limited to, neovascularization associated withtumors.

As used herein, anti-angiogenic treatment or agent refers to anytherapeutic regimen and compound, when used alone or in combination withother treatment or compounds, that can alleviate, reduce, ameliorate,prevent, or place or maintain in a state of remission of clinicalsymptoms or diagnostic markers associated with undesired and/oruncontrolled angiogenesis. Thus, for purposes herein an anti-angiogenicagent refers to an agent that inhibits the establishment or maintenanceof vasculature. Such agents include, but are not limited to, anti-tumoragents, and agents for treatments of other disorders associated withundesirable angiogenesis, such as diabetic retinopathies, restenosis,hyperproliferative disorders and others.

As used herein, non-anti-angiogenic anti-tumor agents refer toanti-tumor agents that do not act primarily by inhibiting angiogenesis.

As used herein, pro-angiogenic agents are agents that promote theestablishment or maintenance of the vasculature. Such agents includeagents for treating cardiovascular disorders, including heart attacksand strokes.

As used herein, undesired and/or uncontrolled angiogenesis refers topathological angiogenesis wherein the influence of angiogenesisstimulators outweighs the influence of angiogenesis inhibitors. As usedherein, deficient angiogenesis refers to pathological angiogenesisassociated with disorders where there is a defect in normal angiogenesisresulting in aberrant angiogenesis or an absence or substantialreduction in angiogenesis.

As used herein, endotheliase refers to a mammalian protein, includinghumans, that has a transmembrane domain and is expressed on the surfaceof endothelial cells and includes a protease domain, particularly anextracellular protease domain, and is preferably a serine protease.Thus, reference, for example, to endotheliase encompasses all proteinsencoded by the endotheliase gene family, or an equivalent moleculeobtained from any other source or that has been prepared syntheticallyor that exhibits the same activity. The endotheliase gene family aretransmembrane proteases expressed in endothelial cells. Endotheliasesare excluded from the MTSPs contemplated herein.

As used herein, the protease domain of an endotheliase refers to thepolypeptide portion of the endotheliase that is the extracellularportion that exhibits protease activity. The protease domain is apolypeptide that includes at least the minimum number of amino acids,generally more than 50 or 100, required for protease activity. Proteaseactivity may be assessed empirically, such as by testing the polypeptidefor its ability to act as a protease. Assays, such as the assaysdescribed in the EXAMPLES, employing a known substrate in place of thetest compounds may be used. Furthermore, since proteases, particularlyserine proteases, have characteristic structures and sequences ormotifs, the protease domain may be readily identified by such structureand sequence or motif.

As used herein, the protease domain of an MTSP protein refers to theprotease domain of an MTSP that is located within or is theextracellular domain of an MTSP and exhibits serine proteolyticactivity. Hence it is at least the minimal portion of the extracellulardomain that exhibits proteolytic activity as assessed by standard assaysin vitro. It refers, herein, to a single chain form heretofore thoughtto be inactive. Exemplary protease domains include at least a sufficientportion of sequences of amino acids set forth as amino acids 615-855 inSEQ ID No. 2 (encoded by nucleotides 1865-2587 in SEQ ID No. 1; see alsoSEQ ID Nos. 49 and 50) from MTSP1, amino acids 205-437 of SEQ ID NO. 4from MTSP3, SEQ ID No. 6, which sets forth the protease domain of MTSP4,and amino acids 217-443 of SEQ ID No. 11 from MTSP6. Also contemplatedare nucleic acid molecules that encode polypeptide that has proteolyticactivity in an in vitro proteolysis assay and that have at least 80%,85%, 90% or 95% sequence identity with the full length of a proteasedomain of an MTSP protein, or that hybridize along their full length toa nucleic acids that encode a protease domain, particularly underconditions of moderate, generally high, stringency.

For each of these protease domains, residues at the N-terminus can becritical for activity, since it has been shown that an Asp in theN-terminus of such proteases is essential for formation of thecatalytically active conformation upon activation cleavage of thezymogen form of the protease. It is shown herein that the proteasedomain of the singles chain form of the protease is catalyticallyactive. Hence the protease domain will require the N-terminal aminoacids; the c-terminus portion may be truncated. The amount that can beremoved can be determined empirically by testing the protein forprotease activity in an in vitro assays that assesses catalyticcleavage.

Hence smaller portions of the protease domains, particularly the singlechain domains, thereof that retain protease activity are contemplated.Such smaller versions will generally be C-terminal truncated versions ofthe protease domains. The protease domains vary in size andconstitution, including insertions and deletions in surface loops. Suchdomains exhibit conserved structure, including at least one structuralfeature, such as the active site triad, primary specificity pocket,oxyanion hole and/or other features of serine protease domains ofproteases. Thus, for purposes herein, the protease domain is a singlechain portion of an MTSP, as defined herein, but is homologous in itsstructural features and retention of sequence of similarity or homologythe protease domain of chymotrypsin or trypsin. Most significantly, thepolypeptide will exhibit proteolytic activity as a single chain.

As used herein, by homologous means about greater than 25% nucleic acidsequence identity, preferably 25% 40%, 60%, 80%, 90% or 95%. The terms“homology” and “identity” are often used interchangeably. In general,sequences are aligned so that the highest order match is obtained (see,e.g.: Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; Carillo et al. (1988) SIAM J Applied Math 48:1073).

By sequence identity, the number of conserved amino acids are determinedby standard alignment algorithms programs, and are used with default gappenalties established by each supplier. Substantially homologous nucleicacid molecules would hybridize typically at moderate stringency or athigh stringency all along the length of the nucleic acid of interest.Also contemplated are nucleic acid molecules that contain degeneratecodons in place of codons in the hybridizing nucleic acid molecule.

Whether any two nucleic acid molecules have nucleotide sequences thatare at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% “identical” can bedetermined using known computer algorithms such as the “FAST A” program,using for example, the default parameters as in Pearson et al. (1988)Proc. Natl. Acad. Sci. USA 85:2444 (other programs include the GCGprogram package (Devereux, J., et al., Nucleic Acids Research 12(I):387(1984)), BLASTP, BLASTN, FASTA (Atschul, S. F., et al., J Molec Biol215:403 (1990); Guide to Huge Computers, Martin J. Bishop, ed., AcademicPress, San Diego, 1994, and Carillo et al. (1988) SIAM J Applied Math48:1073). For example, the BLAST function of the National Center forBiotechnology Information database may be used to determine identity.Other commercially or publicly available programs include, DNAStar“MegAlign” program (Madison, Wis.) and the University of WisconsinGenetics Computer Group (UWG) “Gap” program (Madison Wis.)). Percenthomology or identity of proteins and/or nucleic acid moleucles may bedetermined, for example, by comparing sequence information using a GAPcomputer program (e.g., Needleman et al. (1970) J. Mol. Biol. 48:443, asrevised by Smith and Waterman ((1981) Adv. Appl. Math. 2:482). Briefly,the GAP program defines similarity as the number of aligned symbols(i.e., nucleotides or amino acids) which are similar, divided by thetotal number of symbols in the shorter of the two sequences. Defaultparameters for the GAP program may include: (1) a unary comparisonmatrix (containing a value of 1 for identities and 0 for non-identities)and the weighted comparison matrix of Gribskov et al. (1986) Nucl. AcidsRes. 14:6745, as described by Schwartz and Dayhoff, eds., ATLAS OFPROTEIN SEQUENCE AND STRUCTURE, National Biomedical Research Foundation,pp. 353-358 (1979); (2) a penalty of 3.0 for each gap and an additional0.10 penalty for each symbol in each gap; and (3) no penalty for endgaps.

Therefore, as used herein, the term “identity” represents a comparisonbetween a test and a reference polypeptide or polynucleotide. Forexample, a test polypeptide may be defined as any polypeptide that is90% or more identical to a reference polypeptide. As used herein, theterm at least “90% identical to” refers to percent identities from 90 to99.99 relative to the reference polypeptides. Identity at a level of 90%or more is indicative of the fact that, assuming for exemplificationpurposes a test and reference polynucleotide length of 100 amino acidsare compared. No more than 10% (i.e., 10 out of 100) amino acids in thetest polypeptide differs from that of the reference polypeptides.Similar comparisons may be made between a test and referencepolynucleotides. Such differences may be represented as point mutationsrandomly distributed over the entire length of an amino acid sequence orthey may be clustered in one or more locations of varying length up tothe maximum allowable, e.g. 10/100 amino acid difference (approximately90% identity). Differences are defined as nucleic acid or amino acidsubstitutions, or deletions. At level of homologies or identities aboveabout 85-90%, the result should be independent of the program and gapparameters set; such high levels of identity readily can be assess,often without relying on software.

As used herein, primer refers to an oligonucleotide containing two ormore deoxyribonucleotides or ribonucleotides, preferably more thanthree, from which synthesis of a primer extension product can beinitiated. Experimental conditions conducive to synthesis include thepresence of nucleoside triphosphates and an agent for polymerization andextension, such as DNA polymerase, and a suitable buffer, temperatureand pH.

As used herein, animals include any animal, such as, but are not limitedto, goats, cows, deer, sheep, rodents, pigs and humans. Non-humananimals, exclude humans as the contemplated animal. The MTSPs providedherein are from any source, animal, plant, prokaryotic and fungal.Preferred MTSPs are of animal origin, preferably mammalian origin.

As used herein, genetic therapy involves the transfer of heterologousDNA to the certain cells, target cells, of a mammal, particularly ahuman, with a disorder or conditions for which such therapy is sought.The DNA is introduced into the selected target cells in a manner suchthat the heterologous DNA is expressed and a therapeutic product encodedthereby is produced. Alternatively, the heterologous DNA may in somemanner mediate expression of DNA that encodes the therapeutic product,or it may encode a product, such as a peptide or RNA that in some mannermediates, directly or indirectly, expression of a therapeutic product.Genetic therapy may also be used to deliver nucleic acid encoding a geneproduct that replaces a defective gene or supplements a gene productproduced by the mammal or the cell in which it is introduced. Theintroduced nucleic acid may encode a therapeutic compound, such as agrowth factor inhibitor thereof, or a tumor necrosis factor or inhibitorthereof, such as a receptor therefor, that is not normally produced inthe mammalian host or that is not produced in therapeutically effectiveamounts or at a therapeutically useful time. The heterologous DNAencoding the therapeutic product may be modified prior to introductioninto the cells of the afflicted host in order to enhance or otherwisealter the product or expression thereof Genetic therapy may also involvedelivery of an inhibitor or repressor or other modulator of geneexpression.

As used herein, heterologous DNA is DNA that encodes RNA and proteinsthat are not normally produced in vivo by the cell in which it isexpressed or that mediates or encodes mediators that alter expression ofendogenous DNA by affecting transcription, translation, or otherregulatable biochemical processes. Heterologous DNA may also be referredto as foreign DNA. Any DNA that one of skill in the art would recognizeor consider as heterologous or foreign to the cell in which is expressedis herein encompassed by heterologous DNA. Examples of heterologous DNAinclude, but are not limited to, DNA that encodes traceable markerproteins, such as a protein that confers drug resistance, DNA thatencodes therapeutically effective substances, such as anti-canceragents, enzymes and hormones, and DNA that encodes other types ofproteins, such as antibodies. Antibodies that are encoded byheterologous DNA may be secreted or expressed on the surface of the cellin which the heterologous DNA has been introduced.

Hence, herein heterologous DNA or foreign DNA, includes a DNA moleculenot present in the exact orientation and position as the counterpart DNAmolecule found in the genome. It may also refer to a DNA molecule fromanother organism or species (i.e., exogenous).

As used herein, a therapeutically effective product is a product that isencoded by heterologous nucleic acid, typically DNA, that, uponintroduction of the nucleic acid into a host, a product is expressedthat ameliorates or eliminates the symptoms, manifestations of aninherited or acquired disease or that cures the disease.

As used herein, recitation that a polypeptide consists essentially ofthe protease domain means that the only MTSP portion of the polypeptideis a protease domain or a catalytically active portion thereof. Thepolypeptide may optionally, and generally will, include additionalnon-MTSP-derived sequences of amino acids.

As used herein, cancer or tumor treatment or agent refers to anytherapeutic regimen and/or compound that, when used alone or incombination with other treatments or compounds, can alleviate, reduce,ameliorate, prevent, or place or maintain in a state of remission ofclinical symptoms or diagnostic markers associated with deficientangiogenesis.

As used herein, domain refers to a portion of a molecule, e.g., proteinsor nucleic acids, that is structurally and/or functionally distinct fromother portions of the molecule.

As used herein, protease refers to an enzyme catalyzing hydrolysis ofproteins or peptides. For purposes herein, the protease domain is asingle chain form of an MTSP protein. For MTSP3 and MTSP4 the proteasedomain also includes two chain forms.

As used herein, catalytic activity refers to the activity of the MTSP asa protease as assessed in in vitro proteolytic assays that detectproteolysis of a selected substrate.

As used herein, nucleic acids include DNA, RNA and analogs thereof,including protein nucleic acids (PNA) and mixture thereof. Nucleic acidscan be single or double stranded. When referring to probes or primers,optionally labeled, with a detectable label, such as a fluorescent orradiolabel, single-stranded molecules are contemplated. Such moleculesare typically of a length such that they are statistically unique of lowcopy number (typically less than 5, preferably less than 3) for probingor priming a library. Generally a probe or primer contains at least 14,16 or 30 contiguous of sequence complementary to or identical to a geneof interest. Probes and primers can be 10, 20, 30, 50, 100 or morenucleic acids long.

As used herein, nucleic acid encoding a fragment or portion of an MTSPrefers to a nucleic acid encoding only the recited fragment or portionof MTSP, and not the other contiguous portions of the MTSP.

As used herein, heterologous or foreign DNA and RNA are usedinterchangeably and refer to DNA or RNA that does not occur naturally aspart of the genome in which it is present or which is found in alocation or locations in the genome that differ from that in which itoccurs in nature. Heterologous nucleic acid is generally not endogenousto the cell into which it is introduced, but has been obtained fromanother cell or prepared synthetically. Generally, although notnecessarily, such nucleic acid encodes RNA and proteins that are notnormally produced by the cell in which it is expressed. Any DNA or RNAthat one of skill in the art would recognize or consider as heterologousor foreign to the cell in which it is expressed is herein encompassed byheterologous DNA. Heterologous DNA and RNA may also encode RNA orproteins that mediate or alter expression of endogenous DNA by affectingtranscription, translation, or other regulatable biochemical processes.

As used herein, operative linkage of heterologous DNA to regulatory andeffector sequences of nucleotides, such as promoters, enhancers,transcriptional and translational stop sites, and other signal sequencesrefers to the relationship between such DNA and such sequences ofnucleotides. For example, operative linkage of heterologous DNA to apromoter refers to the physical relationship between the DNA and thepromoter such that the transcription of such DNA is initiated from thepromoter by an RNA polymerase that specifically recognizes, binds to andtranscribes the DNA in reading frame.

As used herein, a sequence complementary to at least a portion of anRNA, with reference to antisense oligonucleotides, means a sequencehaving sufficient complementarily to be able to hybridize with the RNA,preferably under moderate or high stringency conditions, forming astable duplex; in the case of double-stranded MTSP antisense nucleicacids, a single strand of the duplex DNA may thus be tested, or triplexformation may be assayed. The ability to hybridize depends on the degreeof complementarily and the length of the antisense nucleic acid.Generally, the longer the hybridizing nucleic acid, the more basemismatches with a MTSP encoding RNA it can contain and still form astable duplex (or triplex, as the case may be). One skilled in the artcan ascertain a tolerable degree of mismatch by use of standardprocedures to determine the melting point of the hybridized complex.

For purposes herein, conservative amino acid substitutions may be madein any of MTSPs and protease domains thereof provided that the resultingprotein exhibits protease activity. Conservative amino acidsubstitutions, such as those set forth in Table 1, are those that do noteliminate proteolytic activity. Suitable conservative substitutions ofamino acids are known to those of skill in this art and may be madegenerally without altering the biological activity of the resultingmolecule. Those of skill in this art recognize that, in general, singleamino acid substitutions in non-essential regions of a polypeptide donot substantially alter biological activity (see, e.g., Watson et al.Molecular Biology of the Gene, 4th Edition, 1987, The Bejacmin/CummingsPub. co., p. 224). Also included within the definition, is thecatalytically active fragment of an MTSP, particularly a single chainprotease portion. Conservative amino acid substitutions are made, forexample, in accordance with those set forth in TABLE 1 as follows:

TABLE 1 Original residue Conservative substitution Ala (A) Gly; Ser, AbuArg (R) Lys, orn Asn (N) Gln; His Cys (C) Ser Gln (Q) Asn Glu (E) AspGly (G) Ala; Pro His (H) Asn; Gln Ile (I) Leu; Val; Met; Nle; Nva Leu(L) Ile; Val; Met; Nle; Nv Lys (K) Arg; Gln; Glu Met (M) Leu; Tyr; Ile;NLe Val Ornitine Lys; Arg Phe (F) Met; Leu; Tyr Ser (S) Thr Thr (T) SerTrp (W) Tyr Tyr (Y) Trp; Phe Val (V) Ile; Leu; Met; Nle; NvOther substitutions are also permissible and may be determinedempirically or in accord with known conservative substitutions.

As used herein, Abu is 2-aminobutyric acid; Orn is ornithine.

As used herein, the amino acids, which occur in the various amino acidsequences appearing herein, are identified according to theirwell-known, three-letter or one-letter abbreviations. The nucleotides,which occur in the various DNA fragments, are designated with thestandard single-letter designations used routinely in the art.

As used herein, a splice variant refers to a variant produced bydifferential processing of a primary transcript of genomic DNA thatresults in more than one type of mRNA.

As used herein, a probe or primer based on a nucleotide sequencedisclosed herein, includes at least 10, 14, preferably at least 16 or 30or 100 contiguous sequence of nucleotides of SEQ ID Nos. 1, 3, 5, 7, 9or 11.

As used herein, amelioration of the symptoms of a particular disorder byadministration of a particular pharmaceutical composition refers to anylessening, whether permanent or temporary, lasting or transient that canbe attributed to or associated with administration of the composition.

As used herein, antisense polynucleotides refer to synthetic sequencesof nucleotide bases complementary to mRNA or the sense strand of doublestranded

DNA. Admixture of sense and antisense polynucleotides under appropriateconditions leads to the binding of the two molecules, or hybridization.When these polynucleotides bind to (hybridize with) mRNA, inhibition ofprotein synthesis (translation) occurs. When these polynucleotides bindto double stranded DNA, inhibition of RNA synthesis (transcription)occurs. The resulting inhibition of translation and/or transcriptionleads to an inhibition of the synthesis of the protein encoded by thesense strand. Antisense nucleic acid molecule typically contain asufficient number of nucleotides to specifically bind to a targetnucleic acid, generally at least 5 contiguous nucleotides, often atleast 14 or 16 or 30 contiguous nucleotides or modified nucleotidescomplementary to the coding portion of a nucleic acid molecule thatencodes a gene of interest, for example, nucleic acid encoding a singlechain protease domain of an MTSP.

As used herein, an array refers to a collection of elements, such asantibodies, containing three or more members. An addressable array isone in which the members of the array are identifiable, typically byposition on a solid phase support. Hence, in general the members of thearray will be immobilized to discrete identifiable loci on the surfaceof a solid phase.

As used herein, antibody refers to an immunoglobulin, whether natural orpartially or wholly synthetically produced, including any derivativethereof that retains the specific binding ability the antibody. Henceantibody includes any protein having a binding domain that is homologousor substantially homologous to an immunoglobulin binding domain.Antibodies include members of any immunoglobulin claims, including IgG,IgM, IgA, IgD and IgE.

As used herein, antibody fragment refers to any derivative of anantibody that is less then full length, retaining at least a portion ofthe full-length antibody's specific binding ability. Examples ofantibody fragments include,but are not limited to, Fab, Fab′, F(ab)₂,single-chain Fvs (scFV), FV, dsFV diabody and Fd fragments. The fragmentcan include multiple chains linked together, such as by disulfidebridges. An antibody fragment generally contains at least about 50 aminoacids and typically at least 200 amino acids.

As used herein, an Fv antibody fragment is composed of one variableheavy domain (V_(H)) and one variable light domain linked by noncovalentinteractions.

As used herein, a dsFV refers to an Fv with an engineered intermoleculardisulfide bond, which stabilizes the V_(H)—V_(L) pair.

As used herein, an F(ab)₂ fragment is an antibody fragment that resultsfrom digestion of an immunoglobulin with pepsin at pH 4.0-4.5; it may berecombinantly produced.

As used herein, Fab fragments is an antibody fragment that results fromdigestion of an immunoglobulin with papain; it may be recombinantlyproduced.

As used herein, scFVs refer to antibody fragments that contain avariable light chain (VL) and variable heavy chain (VH) covalentlyconnected by a polypeptide linker in any order. The linker is of alength such that the two variable domains are bridged withoutsubstantial interference. Preferred linkers are (Gly-Ser)n residues withsome Glu or Lys residues dispersed throughout to increase solubility.

As used herein, humanized antibodies refer to antibodies that aremodified to include human sequences of amino acids so thatadministration to a human will not provoke an immune response. Methodsfor preparation of such antibodies are known. For example, the hybridomathat expresses the monoclonal antibody is altered by recombinant DNAtechniques to express an antibody in which the amino acid composition ofthe non-variable regions is based on human antibodies. Computer programshave been designed to identify such regions.

As used herein, diabodies are dimeric scFV; diabodies typically haveshorter peptide linkers than scFvs, and they preferentially dimerize.

As used herein, humanized antibodies refer to antibodies that aremodified to include human sequences of amino acids so thatadministration to a human will not provoke an immune response. Methodsfor preparation of such antibodies are known. For example, the hybridomathat expresses the monoclonal antibody is altered by recombinant DNAtechniques to express an antibody in which the amino acid composition ofthe non-variable regions is based on human antibodies. Computer programshave been designed to identify such regions.

As used herein, production by recombinant means by using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA.

As used herein the term assessing is intended to include quantitativeand qualitative determination in the sense of obtaining an absolutevalue for the activity of an MTSP, or a domain thereof, present in thesample, and also of obtaining an index, ratio, percentage, visual orother value indicative of the level of the activity. Assessment may bedirect or indirect and the chemical species actually detected need notof course be the proteolysis product itself but may for example be aderivative thereof or some further substance.

As used herein, biological activity refers to the in vivo activities ofa compound or physiological responses that result upon in vivoadministration of a compound, composition or other mixture. Biologicalactivity, thus, encompasses therapeutic effects and pharmaceuticalactivity of such compounds, compositions and mixtures. Biologicalactivities may be observed in in vitro systems designed to test or usesuch activities. Thus, for purposes herein the biological activity of aluciferase is its oxygenase activity whereby, upon oxidation of asubstrate, light is produced.

As used herein, a combination refers to any association between two oramong more items.

As used herein, a combination refers to any association between two oramong more items. As used herein, a composition refers to any mixture.It may be a solution, a suspension, liquid, powder, a paste, aqueous,non-aqueous or any combination thereof.

As used herein, a conjugate refers to the compounds provided herein thatinclude one or more MTSPs, particularly single chain protease domainsthereof, and one or more targeting agents. These conjugates includethose produced by recombinant means as fusion proteins, those producedby chemical means, such as by chemical coupling, through, for example,coupling to sulfhydryl groups, and those produced by any other methodwhereby at least one MTSP, or a domain thereof, is linked, directly orindirectly via linker(s) to a targeting agent.

As used herein, a targeting agent, is any moiety, such as a protein oreffective portion thereof, that provides specific binding of theconjugate to a cell surface receptor, which, preferably, internalizesthe conjugate or MTSP portion thereof A targeting agent may also be onethat promotes or facilitates, for example, affinity isolation orpurification of the conjugate; attachment of the conjugate to a surface;or detection of the conjugate or complexes containing the conjugate.

As used herein, an antibody conjugate refers to a conjugate in which thetargeting agent is an antibody.

As used herein, humanized antibodies refer to antibodies that aremodified to include human sequences of amino acids so thatadministration to a human will not provoke an immune response. Methodsfor preparation of such antibodies are known. For example, the hybridomathat expresses the monoclonal antibody is altered by recombinant DNAtechniques to express an antibody in which the amino acid composition ofthe non-variable regions is based on human antibodies. Computer programshave been designed to identify such regions.

As used herein, derivative or analog of a molecule refers to a portionderived from or a modified version of the molecule.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, an effective amount of a compound for treating aparticular disease is an amount that is sufficient to ameliorate, or insome manner reduce the symptoms associated with the disease. Such amountmay be administered as a single dosage or may be administered accordingto a regimen, whereby it is effective. The amount may cure the diseasebut, typically, is administered in order to ameliorate the symptoms ofthe disease. Repeated administration may be required to achieve thedesired amelioration of symptoms.

As used herein equivalent, when referring to two sequences of nucleicacids means that the two sequences in question encode the same sequenceof amino acids or equivalent proteins. When equivalent is used inreferring to two proteins or peptides, it means that the two proteins orpeptides have substantially the same amino acid sequence with onlyconservative amino acid substitutions (see, e.g., Table 1, above) thatdo not substantially alter the activity or function of the protein orpeptide. When equivalent refers to a property, the property does notneed to be present to the same extent [e.g., two peptides can exhibitdifferent rates of the same type of enzymatic activity], but theactivities are preferably substantially the same. Complementary, whenreferring to two nucleotide sequences, means that the two sequences ofnucleotides are capable of hybridizing, preferably with less than 25%,more preferably with less than 15%, even more preferably with less than5%, most preferably with no mismatches between opposed nucleotides.Preferably the two molecules will hybridize under conditions of highstringency.

As used herein, an agent that modulates the activity of a protein orexpression of a gene or nucleic acid either decreases or increases orotherwise alters the activity of the protein or, in some manner up- ordown-regulates or otherwise alters expression of the nucleic acid in acell.

As used herein, inhibitor of an the activity of an MTSP encompasses anysubstances that prohibit or decrease production, post-translationalmodification(s), maturation, or membrane localization of the MTSP or anysubstances that interfere with or decrease the proteolytic efficacy ofthereof, particular of a single chain form in vitro.

As used herein, a method for treating or preventing neoplastic diseasemeans that any of the symptoms, such as the tumor, metastasis thereof,the vascularization of the tumors or other parameters by which thedisease is characterized are reduced, ameliorated, prevented, placed ina state of remission, or maintained in a state of remission. It alsomeans that the hallmarks of neoplastic disease and metastasis may beeliminated, reduced or prevented by the treatment. Non-limiting examplesof the hallmarks include uncontrolled degradation of the basementmembrane and proximal extracellular matrix, migration, division, andorganization of the endothelial cells into new functioning capillaries,and the persistence of such functioning capillaries.

As used herein, operatively linked or operationally associated refers tothe functional relationship of DNA with regulatory and effectorsequences of nucleotides, such as promoters, enhancers, transcriptionaland translational stop sites, and other signal sequences. For example,operative linkage of DNA to a promoter refers to the physical andfunctional relationship between the DNA and the promoter such that thetranscription of such DNA is initiated from the promoter by an RNApolymerase that specifically recognizes, binds to and transcribes theDNA. In order to optimize expression and/or in vitro transcription, itmay be necessary to remove, add or alter 5′ untranslated portions of theclones to eliminate extra, potential inappropriate alternativetranslation initiation (i.e., start) codons or other sequences that mayinterfere with or reduce expression, either at the level oftranscription or translation. Alternatively, consensus ribosome bindingsites (see, e.g., Kozak J. Biol. Chem. 266:19867-19870 (1991)) can beinserted immediately 5′ of the start codon and may enhance expression.The desirability of (or need for) such modification may be empiricallydetermined.

As used herein, pharmaceutically acceptable salts, esters or otherderivatives of the conjugates include any salts, esters or derivativesthat may be readily prepared by those of skill in this art using knownmethods for such derivatization and that produce compounds that may beadministered to animals or humans without substantial toxic effects andthat either are pharmaceutically active or are prodrugs.

As used herein, a prodrug is a compound that, upon in vivoadministration, is metabolized or otherwise converted to thebiologically, pharmaceutically or therapeutically active form of thecompound. To produce a prodrug, the pharmaceutically active compound ismodified such that the active compound will be regenerated by metabolicprocesses. The prodrug may be designed to alter the metabolic stabilityor the transport characteristics of a drug, to mask side effects ortoxicity, to improve the flavor of a drug or to alter othercharacteristics or properties of a drug. By virtue of knowledge ofpharmacodynamic processes and drug metabolism in vivo, those of skill inthis art, once a pharmaceutically active compound is known, can designprodrugs of the compound (see, e.g., Nogrady (1985) Medicinal ChemistryA Biochemical Approach, Oxford University Press, New York, pages388-392).

As used herein, a drug identified by the screening methods providedherein refers to any compound that is a candidate for use as atherapeutic or as lead compound for designed a therapeutic. Suchcompounds can be small molecules, including small organic molecules,peptides, peptide mimetics, antisense molecules, antibodies, fragmentsof antibodies, recombinant antibodies and other such compound which canserve as drug candidate or lead compound.

As used herein, production by recombinant means by using recombinant DNAmethods means the use of the well known methods of molecular biology forexpressing proteins encoded by cloned DNA.

As used herein, a promoter region or promoter element refers to asegment of DNA or RNA that controls transcription of the DNA or RNA towhich it is operatively linked. The promoter region includes specificsequences that are sufficient for RNA polymerase recognition, bindingand transcription initiation. This portion of the promoter region isreferred to as the promoter. In addition, the promoter region includessequences that modulate this recognition, binding and transcriptioninitiation activity of RNA polymerase. These sequences may be cis actingor may be responsive to trans acting factors. Promoters, depending uponthe nature of the regulation, may be constitutive or regulated.Exemplary promoters contemplated for use in prokaryotes include thebacteriophage T7 and T3 promoters.

As used herein, a receptor refers to a molecule that has an affinity fora given ligand. Receptors may be naturally-occurring or syntheticmolecules. Receptors may also be referred to in the art as anti-ligands.As used herein, the receptor and anti-ligand are interchangeable.Receptors can be used in their unaltered state or as aggregates withother species. Receptors may be attached, covalently or noncovalently,or in physical contact with, to a binding member, either directly orindirectly via a specific binding substance or linker. Examples ofreceptors, include, but are not limited to: antibodies, cell membranereceptors surface receptors and internalizing receptors, monoclonalantibodies and antisera reactive with specific antigenic determinants[such as on viruses, cells, or other materials], drugs, polynucleotides,nucleic acids, peptides, cofactors, lectins, sugars, polysaccharides,cells, cellular membranes, and organelles.

Examples of receptors and applications using such receptors, include butare not restricted to:

a) enzymes: specific transport proteins or enzymes essential to survivalof microorganisms, which could serve as targets for antibiotic [ligand]selection;

b) antibodies: identification of a ligand-binding site on the antibodymolecule that combines with the epitope of an antigen of interest may beinvestigated; determination of a sequence that mimics an antigenicepitope may lead to the development of vaccines of which the immunogenis based on one or more of such sequences or lead to the development ofrelated diagnostic agents or compounds useful in therapeutic treatmentssuch as for auto-immune diseases

c) nucleic acids: identification of ligand, such as protein or RNA,binding sites;

d) catalytic polypeptides: polymers, preferably polypeptides, that arecapable of promoting a chemical reaction involving the conversion of oneor more reactants to one or more products; such polypeptides generallyinclude a binding site specific for at least one reactant or reactionintermediate and an active functionality proximate to the binding site,in which the functionality is capable of chemically modifying the boundreactant [see, e.g., U.S. Pat. No. 5,215,899];

e) hormone receptors: determination of the ligands that bind with highaffinity to a receptor is useful in the development of hormonereplacement therapies; for example, identification of ligands that bindto such receptors may lead to the development of drugs to control bloodpressure; and

f) opiate receptors: determination of ligands that bind to the opiatereceptors in the brain is useful in the development of less-addictivereplacements for morphine and related drugs.

As used herein, sample refers to anything which may contain an analytefor which an analyte assay is desired. The sample may be a biologicalsample, such as a biological fluid or a biological tissue. Examples ofbiological fluids include urine, blood, plasma, serum, saliva, semen,stool, sputum, cerebral spinal fluid, tears, mucus, amniotic fluid orthe like. Biological tissues are aggregate of cells, usually of aparticular kind together with their intercellular substance that formone of the structural materials of a human, animal, plant, bacterial,fungal or viral structure, including connective, epithelium, muscle andnerve tissues. Examples of biological tissues also include organs,tumors, lymph nodes, arteries and individual cell(s).

As used herein: stringency of hybridization in determining percentagemismatch is as follows:

-   -   1) high stringency: 0.1×SSPE, 0.1% SDS, 65° C.    -   2) medium stringency: 0.2×SSPE, 0.1% SDS, 50° C.    -   3) low stringency: 1.0×SSPE, 0.1% SDS, 50° C.

Those of skill in this art know that the washing step selects for stablehybrids and also know the ingredients of SSPE (see, e.g., Sambrook, E.F. Fritsch, T. Maniatis, in: Molecular Cloning, A Laboratory Manual,Cold Spring Harbor Laboratory Press (1989), vol. 3, p. B.13, see, also,numerous catalogs that describe commonly used laboratory solutions).SSPE is pH 7.4 phophate-buffered 0.18 NaCl. Further, those of skill inthe art recognize that the stability of hybrids is determined by Tm,which is a function of the sodium ion concentration and temperature(Tm=81.5° C.−16.6(log₁₀[Na⁺])+0.41(% G+C)−600/1)), so that the onlyparameters in the wash conditions critical to hybrid stability aresodium ion concentration in the SSPE (or SSC) and temperature.

It is understood that equivalent stringencies may be achieved usingalternative buffers, salts and temperatures. By way of example and notlimitation, procedures using conditions of low stringency are as follows(see also Shilo and Weinberg, Proc. Natl. Acad. Sci. USA, 78:6789-6792(1981)): Filters containing DNA are pretreated for 6 hours at 40° C. ina solution containing 35% formamide, 5×SSC, 50 mM Tris-HCl (pH 7.5), 5mM EDTA, 0.1% PVP, 0.1% Ficoll, 1% BSA, and 500 μg/ml denatured salmonsperm DNA (10×SSC is 1.5 M sodium chloride, and 0.15 M sodium citrate,adjusted to a pH of 7).

Hybridizations are carried out in the same solution with the followingmodifications: 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 μg/ml salmon spermDNA, 10% (wt/vol) dextran sulfate, and 5-20×10⁶ cpm ³²P-labeled probe isused. Filters are incubated in hybridization mixture for 18-20 hours at40° C., and then washed for 1.5 hours at 55° C. in a solution containing2×SSC, 25 mM Tris-HCl (pH 7.4), 5 mM EDTA, and 0.1% SDS. The washsolution is replaced with fresh solution and incubated an additional 1.5hours at 60° C. Filters are blotted dry and exposed for autoradiography.If necessary, filters are washed for a third time at 65-68° C. andreexposed to film. Other conditions of low stringency which may be usedare well known in the art (e.g., as employed for cross-specieshybridizations).

By way of example and not way of limitation, procedures using conditionsof moderate stringency is provided. For example, but not limited to,procedures using such conditions of moderate stringency are as follows:Filters containing DNA are pretreated for 6 hours at 55° C. in asolution containing 6×SSC, 5× Denhart's solution, 0.5% SDS and 100 μg/mldenatured salmon sperm DNA. Hybridizations are carried out in the samesolution and 5-20×10⁶ cpm ³²P-labeled probe is used. Filters areincubated in hybridization mixture for 18-20 hours at 55° C., and thenwashed twice for 30 minutes at 60° C. in a solution containing 1×SSC and0.1% SDS. Filters are blotted dry and exposed for autoradiography. Otherconditions of moderate stringency which may be used are well-known inthe art. Washing of filters is done at 37° C. for 1 hour in a solutioncontaining 2×SSC, 0.1% SDS.

By way of example and not way of limitation, procedures using conditionsof high stringency are as follows: Prehybridization of filterscontaining DNA is carried out for 8 hours to overnight at 65° C. inbuffer composed of 6×SSC, 50 mM Tris-HCl (pH 7.5), 1 mM EDTA, 0.02% PVP,0.02% Ficoll, 0.02% BSA, and 500 μg/ml denatured salmon sperm DNA.Filters are hybridized for 48 hours at 65° C. in prehybridizationmixture containing 100 μg/ml denatured salmon sperm DNA and 5-20×10⁶ cpmof ³²P-labeled probe. Washing of filters is done at 37° C. for 1 hour ina solution containing 2×SSC, 0.01% PVP, 0.01% Ficoll, and 0.01% BSA.This is followed by a wash in 0.1×SSC at 50° C. for 45 minutes beforeautoradiography. Other conditions of high stringency which may be usedare well known in the art.

The term substantially identical or homologous or similar varies withthe context as understood by those skilled in the relevant art andgenerally means at least 70%, preferably means at least 80%, morepreferably at least 90%, and most preferably at least 95% identity.

As used herein, substantially identical to a product means sufficientlysimilar so that the property of interest is sufficiently unchanged sothat the substantially identical product can be used in place of theproduct.

As used herein, substantially pure means sufficiently homogeneous toappear free of readily detectable impurities as determined by standardmethods of analysis, such as thin layer chromatography (TLC), gelelectrophoresis and high performance liquid chromatography (HPLC), usedby those of skill in the art to assess such purity, or sufficiently puresuch that further purification would not detectably alter the physicaland chemical properties, such as enzymatic and biological activities, ofthe substance. Methods for purification of the compounds to producesubstantially chemically pure compounds are known to those of skill inthe art. A substantially chemically pure compound may, however, be amixture of stereoisomers or isomers. In such instances, furtherpurification might increase the specific activity of the compound.

As used herein, target cell refers to a cell that expresses an MTSP invivo.

As used herein, test substance refers to a chemically defined compound(e.g., organic molecules, inorganic molecules, organic/inorganicmolecules, proteins, peptides, nucleic acids, oligonucleotides, lipids,polysaccharides, saccharides, or hybrids among these molecules such asglycoproteins, etc.) or mixtures of compounds (e.g., a library of testcompounds, natural extracts or culture supernatants, etc.) whose effecton an MTSP, particularly a single chain form that includes the proteasedomain or a sufficient portion thereof for activity, as determined by invitro method, such as the assays provided herein.

As used herein, the terms a therapeutic agent, therapeutic regimen,radioprotectant, chemotherapeutic mean conventional drugs and drugtherapies, including vaccines, which are known to those skilled in theart. Radiotherapeutic agents are well known in the art.

As used herein, treatment means any manner in which the symptoms of aconditions, disorder or disease are ameliorated or otherwisebeneficially altered. Treatment also encompasses any pharmaceutical useof the compositions herein.

As used herein, vector (or plasmid) refers to discrete elements that areused to introduce heterologous DNA into cells for either expression orreplication thereof. The vectors typically remain episomal, but may bedesigned to effect integration of a gene or portion thereof into achromosome of the genome. Also contemplated are vectors that areartificial chromosomes, such as yeast artificial chromosomes andmammalian artificial chromosomes. Selection and use of such vehicles arewell known to those of skill in the art. An expression vector includesvectors capable of expressing DNA that is operatively linked withregulatory sequences, such as promoter regions, that are capable ofeffecting expression of such DNA fragments. Thus, an expression vectorrefers to a recombinant DNA or RNA construct, such as a plasmid, aphage, recombinant virus or other vector that, upon introduction into anappropriate host cell, results in expression of the cloned DNA.Appropriate expression vectors are well known to those of skill in theart and include those that are replicable in eukaryotic cells and/orprokaryotic cells and those that remain episomal or those whichintegrate into the host cell genome.

As used herein, protein binding sequence refers to a protein or peptidesequence that is capable of specific binding to other protein or peptidesequences generally, to a set of protein or peptide sequences or to aparticular protein or peptide sequence.

As used herein, epitope tag refers to a short stretch of amino acidresidues corresponding to an epitope to facilitate subsequentbiochemical and immunological analysis of the epitope tagged protein orpeptide. Epitope tagging is achieved by appending the sequence of theepitope tag to the protein-encoding sequence in an appropriateexpression vector. Epitope tagged proteins can be affinity purifiedusing highly specific antibodies raised against the tags.

As used herein, metal binding sequence refers to a protein or peptidesequence that is capable of specific binding to metal ions generally, toa set of metal ions or to a particular metal ion.

As used herein, a composition refers to a any mixture. It may be asolution, a suspension, liquid, powder, a paste, aqueous, non-aqueous orany combination thereof.

As used herein, a combination refers to any association between two oramong more items.

As used herein, fluid refers to any composition that can flow. Fluidsthus encompass compositions that are in the form of semi-solids, pastes,solutions, aqueous mixtures, gels, lotions, creams and other suchcompositions.

As used herein, a cellular extract refers to a preparation or fractionwhich is made from a lysed or disrupted cell.

As used herein, an agent is said to be randomly selected when the agentis chosen randomly without considering the specific sequences involvedin the association of a protein alone or with its associated substrates,binding partners, etc. An example of randomly selected agents is the usea chemical library or a peptide combinatorial library, or a growth brothof an organism.

As used herein, an agent is the to be rationally selected or designedwhen the agent is chosen on a non-random basis which takes into accountthe sequence of the target site and/or its conformation in connectionwith the agent's action. As described in the Examples, there areproposed binding sites for serine protease and (catalytic) sites in theprotein having SEQ ID NO:3 or SEQ ID NO:4. Agents can be rationallyselected or rationally designed by utilizing the peptide sequences thatmake up these sites. For example, a rationally selected peptide agentcan be a peptide whose amino acid sequence is identical to the ATP orcalmodulin binding sites or domains.

For clarity of disclosure, and not by way of limitation, the detaileddescription is divided into the subsections that follow.

B. MTSP PROTEINS, MUTEINS, DERIVATIVES AND ANALOGS THEREOF MTSPs

The MTSPs are a family of transmembrane serine proteases that are foundin mammals and also other species that share a number of commonstructural features including: a proteolytic extracellular C-terminaldomain; a transmembrane domain, with a hydrophobic domain near theN-terminus; a short cytoplasmic domain; and a variable length stemregion containing modular domains. The proteolytic domains sharesequence homology including conserved his, asp, and ser residuesnecessary for catalytic activity that are present in conserved motifs.The MTSPs are synthesized as zymogens, and activated to double chainforms by cleavage. It is shown herein that the single chain proteolyticdomain can function in vitro and, hence is useful in in vitro assays foridentifying agents that modulate the activity of members of this family.Also provided are family members designated MTSP3, MTSP4 and an MTSP6variant.

The MTSP family is a target for therapeutic intervention and also some,may serve as diagnostic markers for tumor development, growth and/orprogression. As discussed, the members of this family are involved inproteolytic processes that are implicated in tumor development, growthand/or progression. This implication is based upon their functions asproteolytic enzymes in processes related to ECM degradative pathways. Inaddition, their levels of expression or level of activation or theirapparent activity resulting from substrate levels or alterations insubstrates and levels thereof differs in tumor cells and non-tumor cellsin the same tissue. Hence, protocols and treatments that alter theiractivity, such as their proteolytic acitivities and roles in signaltransduction, and/or their expression, such as by contacting them with acompound that modulates their activity and/or expression, could impacttumor development, growth and/or progression. Also, in some instances,the level of activation and/or expression may be altered in tumors, suchas lung carcinoma, colon adenocarcinoma and ovarian carcinoma.

The MTSP may serve as a diagnostic marker for tumors. It is shownherein, that MTSP3 and MTSP4 and the MTSP6 variant provided herein areexpressed and/or activated in certain tumors; hence their activation orexpression may serve as a diagnostic marker for tumor development,growth and/or progression. In other instances the MTSP protein canexhibit altered activity by virtue of a change in activity or expressionof a co-factor therefor or a substrate therefor. In addition, in someinstances, these MTSPS and/or variants thereof may be shed from cellsurfaces. Detection of the shed MTSPS, particularly the extracellulardomains, in body fluids, such as serum, blood, saliva, cerebral spinalfluid, synovial fluid and interstitial fluids, urine, sweat and othersuch fluids and secretions, may serve as a diagnostic tumor marker. Inparticular, detection of higher levels of such shed polypeptides in asubject compared to a subject known not to have any neoplastic diseaseor compared to earlier samples from the same subject, can be indicativeof neoplastic disease in the subject.

Provided herein are isolated substantially pure single polypeptides thatcontain the protease domain of an MTSP as a single chain. The MTSPscontemplated herein are not expressed on endothelial cells, and,preferably, are expressed on tumor cells, typically at a level thatdiffers from the level in which they are expressed in the non-tumor cellof the same type. Hence, for example, if the MTSP is expressed in anovarian tumor cell, to be of interest herein with respect to ovariancancer, it is expressed at the same level in non-tumor ovarian cells.MTSP protease domains include the single chain protease domains ofMTSP1, MTSP3, MTSP4, MTSP6 and other such proteases, including, but arenot limited to, corin, enterpeptidase, human airway trypsin-likeprotease (HAT), MTSP1, TMPRS2, and TMPRSS4.

Provided are the protease domains or proteins that include a portion ofan MTSP that is the protease domain of any MTSP, particularly an MTSP1,MTSP3, MTSP4 and MTSP6. The protein can also include other non-MTSPsequences of amino acids, but will include the protease domain or asufficient portion thereof to exhibit catalytic activity in any in vitroassay that assess such protease activity, such as any provided herein.

Also provided herein are nucleic acid molecules that encode MTSPproteins and the encoded proteins. In particular, nucleic acid moleculesencoding MTSP-3 and MTSP-4 from animals, including splice variantsthereof are provided. The encoded proteins are also provided. Alsoprovided are functional domains thereof.

In specific aspects, the MTSP protease domains, portions thereof, andmuteins thereof are from or based on animal MTSPS, including, but arenot limited to, rodent, such as mouse and rat; fowl, such as chicken;ruminants, such as goats, cows, deer, shee; ovine, such as pigs; andhumans.

In particular, MTSP derivatives can be made by altering their sequencesby substitutions, additions or deletions that provide for functionallyequivalent molecules. Due to the degeneracy of nucleotide codingsequences, other nucleic sequences which encode substantially the sameamino acid sequence as a MTSP gene can be used. These include but arenot limited to nucleotide sequences comprising all or portions of MTSPgenes that are altered by the substitution of different codons thatencode the amino acid residue within the sequence, thus producing asilent change. Likewise, the MTSP derivatives include, but are notlimited to, those containing, as a primary amino acid sequence, all orpart of the amino acid sequence of MTSP, including altered sequences inwhich functionally equivalent amino acid residues are substituted forresidues within the sequence resulting in a silent change. For example,one or more amino acid residues within the sequence can be substitutedby another amino acid of a similar polarity which acts as a functionalequivalent, resulting in a silent alteration. Substitutes for an aminoacid within the sequence may be selected from other members of the classto which the amino acid belongs. For example, the nonpolar (hydrophobic)amino acids include alanine, leucine, isoleucine, valine, proline,phenylalanine, tryptophan and methionine. The polar neutral amino acidsinclude glycine, serine, threonine, cysteine, tyrosine, asparagine, andglutamine. The positively charged (basic) amino acids include arginine,lysine and histidine. The negatively charged (acidic) amino acidsinclude aspartic acid and glutamic acid (see, e.g., Table 1).

In a preferred embodiment, the substantially purified MTSP protease isencoded by a nucleic acid that hybridizes to the a nucleic acid moleculecontaining the protease domain encoded by the nucleotide sequence setforth in any of SEQ. ID Nos. 1, 3, 5, 7, 9 or 11 under at leastmoderate, generally high, stringency conditions, such that the proteasedomain encoding nucleic acid thereof hybridizes along its full length.In preferred embodiments the substantially purified MTSP protease is asingle chain polypeptide that includes substantially the sequence ofamino acids set forth in any SEQ ID Nos. 2, 4, 6, 8, 10 and 12 thatencodes the protease domain. Specific sequences for the following humanMTSPs and domains thereof are provided as follows: SEQ ID No. 3 MTSP3nucleic acid sequence; SEQ ID No. 4 MTSP3 amino acid sequence; SEQ IDNo. 5 MTSP4 nucleic acid encoding the protease domain; SEQ ID No. 6MTSP4 amino acid sequence of the protease domain; SEQ ID No. 7 MTSP4-Lnucleic acid sequence; SEQ ID No. 8 MTSP4-L amino acid sequence; SEQ IDNo. 9 MTSP4-S nucleic acid sequence; SEQ ID No. 10 MTSP4-S amino acidsequence; SEQ ID No. 11 MTSP6 nucleic acid sequence; SEQ ID No. 12 MTSP6amino acid sequence. SEQ ID No. 1 sets forth the nucleic acid sequenceof the long form of MTSP1; SEQ ID No. 2 the encoded amino acid sequence;SEQ ID No. 49 sets forth the sequence of a protease domain of an MTSP1,and SEQ ID No. 50 the sequence of the encoded single chain proteasedomain thereof. FIGS. 1-3 depict the structural organization of theMTSP3, MTSP4 and MTSP6, respectively.

In particular, exemplary protease domains include at least a sufficientportion of sequences of amino acids set forth as amino acids 615-855 inSEQ ID No. 2 (encoded by nucleotides 1865-2587 in SEQ ID No. 1; see alsoSEQ ID Nos. 49 and 50) from MTSP1 (matriptase), amino acids 205-437 ofSEQ ID NO. 4 from MTSP3, SEQ ID No. 6, which sets forth the proteasedomain of MTSP4, and amino acids 217-443 of SEQ ID No. 11 from MTSP6.

Also contemplated are nucleic acid molecules that encode a single chainMTSP protease that have proteolytic activity in an in vitro proteolysisassay and that have at least 60%, 70%, 80%, 85%, 90% or 95% sequenceidentity with the full length of a protease domain of an MTSP protein,or that hybridize along their full length to a nucleic acids that encodea protease domain, particularly under conditions of moderate, generallyhigh, stringency. As above, the encoded polypeptides contain theprotease as a single chain.

The isolated nucleic acids may include of at least 8 nucleotides of anMTSP sequence. In other embodiments, the nucleic acids may contain least25 (continuous) nucleotides, 50 nucleotides, 100 nucleotides, 150nucleotides, or 200 nucleotides of a MTSP sequence, or a full-lengthMTSP coding sequence. In another embodiment, the nucleic acids aresmaller than 35, 200 or 500 nucleotides in length. Nucleic acids can besingle or double stranded. Nucleic acids that hybridizes to orcomplementary to the foregoing sequences, in particular the inversecomplement to nucleic acids that hybridizes to the foregoing sequences(i.e., the inverse complement of a nucleic acid strand has thecomplementary sequence running in reverse orientation to the strand sothat the inverse complement would hybridize without mismatches to thenucleic acid strand; thus, for example, where the coding strand is thathybridizes to a nucleic acid with no mismatches between the codingstrand and the that hybridizes strand, then the inverse complement ofthe that hybridizes strand is identical to the coding strand) are alsoprovided. In specific aspects, nucleic acids are provided that include asequence complementary to (specifically are the inverse complement of)at least 10, 25, 50, 100, or 200 nucleotides or the entire coding regionof an MTSP encoding nucleic acid, particularly the protease domainthereof. For MTSP3 and MTSP4 the full-length protein or domain or activefragment thereof.

For each of the nucleic acid molecules, the nucleic acid can be DNA orRNA or PNA or other nucleic acid analogs or may include non-naturalnucleotide bases.

Also provided are isolated nucleic acid molecules that include asequence of nucleotides complementary to the nucleotide sequenceencoding an MTSP.

Probes and primers derived from the nucleic acid molecules are provided,

Such probes and primers contain at least 8, 14, 16, 30, 100 or morecontiguous nucleotides with identity to contiguous nucleotides of anMTSP, including, but are not limited to, MTSP1, MTSP3, MTSP4 and MTSP6.The probes and primers are optionally labelled with a detectable label,such as a radiolabel or a fluorescent tag, or can be mass differentiatedfor detection by mass spectrometry or other means.

Plasmids and vectors containing the nucleic acid molecules are alsoprovided. Cells containing the vectors, including cells that express theencoded proteins are provided. The cell can be a bacterial cell, a yeastcell, a fungal cell, a plant cell, an insect cell or an animal cell.Methods for producing an MTSP or single chain form of the proteasedomain thereof by, for example, growing the cell under conditionswhereby the encoded MTSP is expressed by the cell, and recovering theexpressed protein, are provided herein. As noted, for MTSP3 and MTSP4,the full-length zymogens and activated proteins and activated (twostrand) protease and single chain protease domains are provided.

Except for the MTSP proteins (MTSP3 and MTSP4) heretofore unidentifiedand provided herein, the isolated polypeptides contain the MTSP proteasedomain or a catalytically active portion thereof and, generally, do notcontain additional MTSP. Hence isolated, substantially pure proteases,protease domains or catalytically active portion thereof in single chainform of MTSPs are provided. The protease domains may be included in alonger protein, but such longer protein is not the MTSP zymogen.

Thus, MTSP3 and MTSP4 proteins are provided. For these proteins, thedomains, fragments, derivatives or analogs that are functionally active,i.e., capable of exhibiting one or more functional activities associatedwith the MTSP protein, e.g., serine protease activity, immunogenicityand antigenicity, are provided. As discussed above, the protease domainsthereof are also provided. For MTSP3 and MTSP4, the zymogens andactivated forms, and also, the single chain and double chain, activatedprotease domains are provided.

Also provided are nucleic acid molecules that hybridize to theabove-noted sequences of nucleotides encoding MTSP3 and MTSP4 (SEQ IDNos. 3, 5, 7 and 9) at least at low stringency, more preferably atmoderate stringency, and most preferably at high stringency, and thatencode the protease domain and/or the full length protein or otherdomains of an MTSP family member, such as MTSP3, MTSP4, MTSP6 or asplice variant or allelic variant thereof, or MTSP6 or a splice variantor allelic variant thereof. Preferably the molecules hybridize undersuch conditions along their full length for at least one domain andencode at least one domain, such as the protease or extracellulardomain, of the polypeptide. In particular, such nucleic acid moleculesinclude any isolated nucleic fragment that encodes at least one domainof a membrane serine protease, that (1) contains a sequence ofnucleotides that encodes the protease or a domain thereof, and (2) isselected from among:

-   -   (a) a sequence of nucleotides that encodes the protease or a        domain thereof includes a sequence of nucleotides set forth        above;    -   (b) a sequence of nucleotides that encodes such portion or the        full length protease and hybridizes under conditions of high        stringency, preferably to nucleic acid that is complementary to        a mRNA transcript present in a mammalian cell that encodes such        protein or fragment thereof;    -   (c) a sequence of nucleotides that encodes a transmembrane        protease or domain thereof that includes a sequence of amino        acids encoded by such portion or the full length open reading        frame; and    -   (d) a sequence of nucleotides that encodes the transmembrane        protease that includes a sequence of amino acids encoded by a        sequence of nucleotides that encodes such subunit and hybridizes        under conditions of high stringency to DNA that is complementary        to the mRNA transcript.

Exemplary MTSPs

The above discussion provides an overview and some details of theexemplified MTSPs. The following discussion provides additional details(see, also, EXAMPLES).

MTSP1 (Matriptase)

Matriptase is a trypsin-like serine protease with broad spectrumcleavage activity and two potential regulatory modules. It was named“matriptase” because its ability to degrade the extra-cellular matrixand its trypsin-like activity. When isolated from breast cancer cells(or T-47D cell conditioned medium), matriptase has been reported to beprimarily in an uncomplexed form. Matriptase has been isolated fromhuman milk; when isolated from human milk, matriptase was reported to bein one of two complexed forms, 95 kDa (the predominant form) and 110kDa; uncomplexed matriptase was not detected. (Liu, et al., J. Biol.Chem. 274(26):18237-18242 (1999).) It has been proposed that matriptaseexists as an uncomplexed protease when in its active state. In breastmilk, matriptase has been reported to exist in complex with a fragmentof hepatocyte growth factor inhibitor-1 (HAI-1), a Kuntz-type serineprotease inhibitor having activity against trypsin-like serineproteases.

Ecotin and Ecotin M84R/M85R are macromolecular inhibitors of serineproteases of the chymotrypsin fold and inhibit ductal branching,morphogenesis and differentiation of the explanted ductal prostate. PC-3is a cell line derived from prostate cancer epithelial cells. Ecotin andM84R/M85R ecotin were found to decrease tumor size and metastasis inPC-3 implanted nude mice.

Matriptase has been isolated and its encoding nucleic acids cloned fromT-47D human breast cancer cell-conditioned medium (Lin et al. (1999) J.Biol. Chem. 274:18231-18236). Upon analysis of the cDNA, it wasdetermined that the full length protease has 683 amino acids andcontains three main structural regions: a serine protease domain nearthe carboxyl-terminal region, four tandem low-density lipoproteinreceptor domains, and two tandem complement subcomponents C1r and C1s.

Studies to identify additional serine proteases made by cancer cellswere done using PC-3 cells. A serine protease termed “MT-SP1”, reportedto be a transmembrane protease was cloned (Takeuchi et al. (1999) Proc.Natl. Acad. Sci. U.S.A. 96:11054-11061). It was subsequently found theoriginally identified matriptase sequence is included in the translatedsequence of the cDNA that encodes MT-SP1. The matriptase cDNA wasreported to be a partial MT-SP1 cDNA and to lack 516 of the codingnucleotides (Takeuchi, et al., J. Biol. Chem 275:26333-26342 (2000).)Since the reported matriptase encoding cDNA sequence encoded a possibleinitiating methionine, it was proposed that alternative splicing couldyield a protein lacking the N-terminal region of MTSP1.

Matriptase and MT-SP1 demonstrate trypsin-like protease activity and areType II transmembrane proteins with a common extracellular proteasedomain. Studies of substrate specificity of MT-SP1 reveal thatprotease-activated receptor 2

(PAR2) and single-chain urokinase-type plasminogen activator (sc-uPA)are macromolecular substrates of MT-SP1. PAR2 is functions ininflammation, cytoprotection and/or cell adhesion, while sc-uPa isfunctions in tumor cell invasion and metastasis.

An exemplary nucleotide sequences encoding a human MTSP1 is set forth inSEQ ID Nos 1 and 2 (see also SEQ ID Nos. 49 and 50 for the proteasedomain thereof). As previously noted SEQ ID No. 1 sets for anMTSP1-encoding nucleic acid sequence. This sequence is the longerversion and includes the protease domain, which is common to bothvariants Nucleic acids encoding the MTSP that hybridizes to thenucleotide sequence set forth in SEQ ID No. 1 can be obtained by anymethod known in the art, e.g, by PCR amplification using syntheticprimers that hybridizes to the 3′ and 5′ ends of the sequence and/or bycloning from a cDNA or genomic library using a PCR amplification productor an oligonucleotide specific for the gene sequence (e.g., as describedin Section C herein). Homologs (e.g., nucleic acids of the above-listedgenes of species other than human) or other related sequences (e.g.,paralogs) and muteins can be obtained by low, moderate or highstringency hybridization with all or a portion of the particularsequence provided as a probe using methods well known in the art fornucleic acid hybridization and cloning.

Isolated single chain protease domains of MTSP1 proteins from animalsare provided herein. As shown herein, the single chain protease domainis catalytically active and can be used in a variety of drug screeningassays, particularly in vitro proteolytic assays. Exemplary MTSPprotease domains are set forth as the amino acids (615-855 of SEQ ID No.2) encoded by nucleotides 1865-2587 of SEQ ID No. 1 (see, also, SEQ IDNos. 49 and 50). The MTSP1 single chain protease domain is catalyticallyactive

Muteins of the MTSP1 proteins are provided. In the activated doublechain molecule, residue 731 forms a disulfide bond with the Cys atresidue 604. In the single chain form, the residue at 731 in theprotease domain is free. Muteins in which Cys residues, particularly thefree Cys residue (amino acid 731 in SEQ ID No. 2) in the single chainprotease domain are provided. Other muteins in which conservative aminoacids replacements are effected and that retain proteolytic activity asa single chain are also provided. Such changes may be systematicallyintroduced and tested for activity in in vitro assays, such as thoseprovided herein.

MTSP3

In a specific embodiment, a nucleic acid that encodes a MTSP, designatedMTSP3 is provided. In particular, the nucleic acid includes an openreading frame within the following sequence of nucleotides set forth inSEQ ID No. 3. In particular the protein is encoded by the open readingframe that begins at nucleotide 261 and ends at 1574.

Also provided are nucleic acid molecules that hybridize under conditionsof at least low stringency, preferably moderate stringency, morepreferably high stringency to the following sequence of nucleic acids(SEQ ID No. 3), particularly to the open reading frame encompassed bynucleotides that encode a single protease domain thereof, or any domainof MTSP3

Also included are substantially purified MTSP3 zymogen, activated doublechains, single chain protease domains and double chain protease domains.These are encoded by a nucleic acid that includes sequence encoding aprotease domain that exhibits proteolytic activity and that hybridizesto a nucleic acid molecule having a nucleotide sequence set forth in SEQID No. 3, typically under moderate, generally under high stringency,conditions and most preferably along the full length of the proteasedomain. Splice variants are also contemplated herein.

In a preferred embodiment, the isolated nucleic acid fragment hybridizesto the nucleic acid having the nucleotide sequence set forth in SEQ IDNo: 3 under high stringency conditions, and preferably comprises thesequence of nucleotides set forth in any of SEQ ID Nos. 3 or comprises aportion thereof that encodes a transmembrane domain and may additionallyinclude a LDLR domain, a scavenger-receptor cysteine rich (SRCR) domainand a serine protease catalytic domain or any other identified domain(see FIGURES) or comprises nucleic acid molecule that encodes theprotein encoded by SEQ ID NO. 4.

The isolated nucleic acid fragment is DNA, including genomic or cDNA, oris RNA, or can include other components, such as protein nucleic acid.The isolated nucleic acid may include additional components, such asheterologous or native promoters, and other transcriptional andtranslational regulatory sequences, these genes may be linked to othergenes, such as reporter genes or other indicator genes or genes thatencode indicators.

Also provided is an isolated nucleic acid molecule that includes thesequence of molecules that is complementary to the nucleotide sequenceencoding the MTSP or the portion thereof.

Also provided are fragments thereof that can be used as probes orprimers and that contain at least about 10 nucleotides, more preferably14 nucleotides, more preferably at least about 16 nucleotides, mostpreferably at least about 30 nucleotides.

Hence provided herein are polypeptides that are encoded by such nucleicacid molecules. Included among those polypeptides are the MTSP3 proteasedomain or a polypeptide with conservative amino acid changes such thatthe specificity and protease activity remains substantially unchange. Inparticular, a substantially purified mammalian MTSP protein is providedthat has a transmembrane domain and may additionally include a CUBdomain, one or more of an LDLR domain(s), a scavenger-receptor cysteinerich (SRCR) domain and a serine protease catalytic domain is provided.

Also provided is a substantially purified protein comprising a sequenceof amino acids that has at least 60%, more preferably at least about90%, most preferably at least about 95%, identity to the MTSP3, whereinthe percentage identity is determined using standard algorithms and gappenalties that maximize the percentage identity. The human MTSP3 proteinis most preferred, although other mammalian MTSP3 proteins arecontemplated.

Muteins of MTSP3, particularly those in which Cys residues, such as theCys310 in the single chain protease domain, is replaced with anotheramino acid that does not eliminate the activity, are provided.

MTSP4

Among the proteins provided herein is MTSP4. MTSP4 is highly expressedin the liver, and is expressed in substantially lower levels in othertissues (see, EXAMPLES). It is also expressed in non-liver-derivedtumors (see EXAMPLES), including Burkitt's lymphoma, colorectaladenocarcinoma (SW480), lung carcinoma (A549), and in leukemic cells,indicating a role in one or more of tumor progression, tumor invasion,tumor growth and tumor metastases.

Also provided are nucleic acid molecules that hybridize under conditionsof at least low stringency, preferably moderate stringency, morepreferably high stringency to the sequence of nucleic acids set forth inSEQ ID Nos. 5, 7 or 9), particularly to the open reading frameencompassed by nucleotides that encode a single protease domain thereof,or any domain of an MTSP4.

Also included are substantially purified MTSP4 zymogens, activateddouble chains, single chain protease domains and double chain proteasedomains. These are encoded by a nucleic acid that includes sequenceencoding a protease domain that exhibits proteolytic activity and thathybridizes to a nucleic acid molecule having a nucleotide sequence setforth in SEQ ID Nos. 5, 7 and 9, typically under moderate, generallyunder high stringency, conditions and most preferably along the fulllength of the protease domain.

In a preferred embodiment, the isolated nucleic acid fragment hybridizesto the nucleic acid having the nucleotide sequence set forth in SEQ IDNo: 5, 7 or 9 under high stringency conditions, and preferably comprisesthe sequence of nucleotides set forth in any of SEQ ID Nos. 5, 7 or 9comprises a portion thereof that encodes a transmembrane domain and mayadditionally include a LDLR domain, a scavenger-receptor cysteine rich(SRCR) domain and a serine protease catalytic domain or any otheridentified domain (see FIGURES) or comprises nucleic acid molecule thatencodes the protein encoded by SEQ ID NO. 6, 9 or 10..

The isolated nucleic acid fragment is DNA, including genomic or cDNA, oris RNA, or can include other components, such as protein nucleic acid.The isolated nucleic acid may include additional components, such asheterologous or native promoters, and other transcriptional andtranslational regulatory sequences, these genes may be linked to othergenes, such as reporter genes or other indicator genes or genes thatencode indicators.

Also provided is an isolated nucleic acid molecule that includes thesequence of molecules that is complementary to the nucleotide sequenceencoding and MTSP4 or the portion thereof.

Also provided are fragments thereof that can be used as probes orprimers and that contain at least about 10 nucleotides, more preferably14 nucleotides, more preferably at least about 16 nucleotides, mostpreferably at least about 30 nucleotides.

In particular nucleic acid molecules encoding two forms of MTSP4 areprovide. The encoded proteins are multi-domain, type II membrane-typeserine proteases and include a transmembrane domain at the N terminusfollowed by a CUB domain, 3 LDLR domains and a trypsin-like serineprotease domain at the C terminus. The difference between the two forms,which are splice variants, is the absence in MTSP4-S of a 432-bpnucleotide sequence between the transmembrane and the CUB domains (seeFIG. 2; see, also SEQ ID Nos. 5-10).

Also provided is a nucleic acid that encodes the extracellular proteasedomain of an MTSP4 is provided. Both forms of MTSP4 exemplified hereininclude a protease domain in common (see SEQ ID Nos. 5 and 6).

In particular, the extracellular protease domain of the MTSP4 proteinsis encoded by the open reading frame that begins at nucleotide 1 andends at 708 (TGA) (SEQ ID No. 5. This open reading frame encodes aportion of the MSTP4 protein and includes the protease domain. Fulllength MSTP4 proteins (SEQ ID Nos. 7 and 9) include the above domain.The extracellular protease domain, as a single chain, and also anactivated double chain, exhibit protease activity. The disulfide bondsthat form that two chain form of MTSP forms are likely between Cys415and Cys535 for MTSP4-S, and between Cys559 and Cys679 for MTSP4-L.

For use of the single chain protease domain thereof, it is of interestto replace the free Cys (i.e. Cys535 (Cys679)) in the protease domainwith another amino acid, such as any amino acid that does not alter thefunction (such change is likely to be any amino acid). Thus, muteins ofMTSP4, particularly those in which Cys residues, such as the Cys535 andCys679 in the single chain protease domains of MTSP4-S and MTSP4-L,respectively, are provided.

MTSP6

Nucleic acid and the encoded MTSP6 protein of an exemplary MTSP6 arealso provided. The respective sequences are set forth in SEQ ID Nos. 11and 12. The MTSP6 DNA and protein sequences were analyzed using DNAStrider (version 1.2). The ORF encoding the MTSP6 variant providedherein is composed of 1,362 bp, which translate into a 453-amino acidprotein. MTSP6 is a multi-domain, type-II membrane-type serine proteasecontaining a transmembrane domain (amino acids 48-68) at the N-terminusfollowed by a LDLRa domain (LDL receptor domain class a) (amino acids72-108), a SR domain (Scavenger receptor Cys-rich domain)(amino acids109-205), and a trypsin-like serine protease domain (amino acids216-443) (see FIG. 3). Muteins of MTSP6, particularly those in which Cysresidues, such as the

Cys324 in the single chain protease domain of MTSP6 are provided.

International PCT application No. WO 00/52044 describes MTSPs thatresemble the MTSP6 provided herein. The polypeptide provided thereindiffers at single amino acid positions, such as 90 in SEQ ID No. 12 (Alais replaced with a Thr), and significantly from the instant MTSP6 inthat ten amino acids (amino acid nos. 46-55 in SEQ ID No. 12) arereplaced with the eleven amino acids: phe glu val phe ser gln ser serser leu gly (SEQ ID No. 59) resulting in a protein that is one 454 aminoacids long.

There are a few other amino acid sequence differences and a number ofnucleic acid sequence differences. Significantly, there are substantialdifferences in the protease domain at amino acids 368-394 (368ICNHRDVYGGIISPSMLCAGYLTGGVD—394; SEQ ID No. 12) are replaced at position369-396 with animo acids: 369 DLQPQ—GRVRWHHLPLHALRGLPDGWRWN 396, wherethe differences from 368-394 (Seq ID No. 12) are indicated.

In addition, a second C-terminus truncated variant with an alteredprotease domain is identified in the PCT application. The variant is thesame as the 454 variant through amino acid 261 thereof (corresponding to160 of SEQ ID No. 12 herein), followed by 33 amino acids (see SEQ ID No.60 herein) that differ by virtue of a frame shift.

C. TUMOR SPECIFICITY AND TISSUE EXPRESSION PROFILES

Each MTSP has a characteristic tissue expression profile; the MTSPs inparticular, although not exclusively expressed or activated in tumors,exhibit characteristic tumor tissue expression or activation profiles.In some instances, MTSPs may have different activity in a tumor cellfrom a non-tumor cell by virtue of a change in a substrate or cofactorthereof or other factor that would alter the apparent functionalactivity of the MTSP. Hence each can serve as a diagnostic marker forparticular tumors, by virtue of a level of activity and/or expression orfunction in a subject (i.e. a mammal, particularly a human) withneoplastic disease, compared to a subject or subjects that do not havethe neoplastic disease. In addition, detection of activity (and/orexpression) in a particular tissue can be indicative of neoplasticdisease. Shed MTSPs in body fluids can be indicative of neoplasticdisease. Also, by virtue of the activity and/or expression profiles ofeach, they can serve as therapeutic targets, such as by administrationof modulators of the activity thereof, or, as by administration of aprodrug specifically activated by one of the MTSPs.

Tissue Expression Profiles

MTSP3

The MTSP3 transcript was detected in lung carcinoma (LX-1), colonadenocarcinoma (CX-1), colon adenocarcinoma (GI-112) and ovariancarcinoma (GI-102). No apparent signal was detected in another form oflung carcinoma (GI-117), breast carcinoma (GI-101), pancreaticadenocarcinoma (GI-103) and prostatic adenocarcinoma (PC3).

MTSP1 is expressed in breast cancers.

MTSP4

The MTSP4 transcript, a DNA fragment encoding part of the LDL receptordomain and the protease domain was used to probe an RNA blot composed of76 different human tissues (catalog number 7775-1; human multiple tissueexpression (MTE) array; CLONTECH). As in the northern analysis of gelblot, a very strong signal was observed in the liver. Signals in othertissues were observed in (decreasing signal level): fetalliver>heart=kidney=adrenal gland=testis=fetal heart and kidney=skeletalmuscle=bladder=placenta>brain=spinal cord=colon=stomach=spleen=lymphnode=bone marrow=trachea=uterus=pancreas=salivary gland=mammarygland=lung. MTSP4 is also expressed less abundantly in several tumorcell lines including HeLa S3=leukemia K-562=Burkitt's lymphomas (Rajiand Daudi)=colorectal adenocarcinoma (SW480)>lung carcinoma(A549)=leukemia MOLT-4=leukemia HL-60. PCR of the MTSP4 transcript fromcDNA libraries made from several human primary tumors xenografted innude mice (human tumor multiple tissue cDNA panel, catalog numberK1522-1, CLONTECH) was performed using MTSP4-specific primers. The MTSP4transcript was detected in breast carcinoma (GI-101), lung carcinoma(LX-1), colon adenocarcinoma (GI-112) and pancreatic adenocarcinoma(GI-103). No apparent signal was detected in another form of lungcarcinoma (GI-117), colon adenocarcinoma (CX-1), ovarian carcinoma(GI-102), and prostatic adenocarcinoma (PC3). The MTSP4 transcript wasalso detected in LNCaP and PC-3 prostate cancer cell lines as well as inHT-1080 human fibrosarcoma cell line.

Gene Expression Profile of MTSP6 in Normal and Tumor Tissues

To obtain information regarding the gene expression profile of the MTSP6transcript, a 495 by DNA fragment obtained from PCR reaction withprimers Ch17-NSP-3 and NSP-4AS was used to probe an RNA blot composed of76 different human tissues (catalog number 7775-1; human multiple tissueexpression (MTE) array; CLONTECH). The strongest signal was observed induodenum. Signal in other tissues were observed in (decreased signallevel): Stomach>trachea=mammary gland=thyroid gland=salivarygland=pituitarygland=pancreas>kidney>lung>jejunum=ileum=ilocecum=appendix=fetalkidney>fetal lung. Very weak signals can also be detected in severalother tissues.

MTSP6 is also expressed in several tumor cell lines including HeLaS3>colorectal adenocarcinoma (SW480)>leukemia MOLT-4>leukemia K-562. PCRanalysis of the MTSP6 transcript from cDNA libraries made from severalhuman primary tumors xenografted in nude mice (human tumor multipletissue cDNA panel, catalog number K1522-1, CLONTECH) was performed usingMTSP6-specific primers (Ch17-NSP-3 and Ch17-NSP2AS). The MTSP6transcript was strongly detected in lung carcinoma (LX-1), moderatelydetected in pancreatic adenocarcinoma (GI-103), weakly detected inovarian carcinoma (GI-102); and very weakly detected in colonadenocarcinoma (GI-112 and CX-1), breast carcinoma (GI-101), lungcarcinoma (GI-117) and prostatic adenocarcinoma (PC3). The MTSP6transcript was also detected in breast cancer cell line MDA-MB-231,prostate cancer cell line PC-3, but not in HT-1080 human fibrosarcomacell line. MTSP6 is also expressed in mammary gland carcinoma cDNA(Clontech). MTSP6 is also over expressed in ovarian tumor cells.

D. IDENTIFICATION AND ISOLATION OF MTSP PROTEIN GENES

The MTSP proteins, or domains thereof, can be obtained by methods wellknown in the art for protein purification and recombinant proteinexpression. Any method known to those of skill in the art foridentification of nucleic acids that encode desired genes may be used.Any method available in the art can be used to obtain a full length(i.e., encompassing the entire coding region) cDNA or genomic DNA cloneencoding an MTSP protein. In particular, the polymerase chain reaction(PCR) can be used to amplify a sequence identified as beingdifferentially expressed in normal and tumor cells or tissues, e.g.,nucleic acids encoding an MTSP protein (SEQ. NOs: 1-12), in a genomic orcDNA library. Oligonucleotide primers that hybridize to sequences at the3′ and 5′ termini of the identified sequences can be used as primers toamplify by PCR sequences from a nucleic acid sample (RNA or DNA),preferably a cDNA library, from an appropriate source (e.g., tumor orcancer tissue).

PCR can be carried out, e.g., by use of a Perkin-Elmer Cetus thermalcycler and Taq polymerase (Gene Amp™). The DNA being amplified caninclude mRNA or cDNA or genomic DNA from any eukaryotic species. One canchoose to synthesize several different degenerate primers, for use inthe PCR reactions. It is also possible to vary the stringency ofhybridization conditions used in priming the PCR reactions, to amplifynucleic acid homologs (e.g., to obtain MTSP protein sequences fromspecies other than humans or to obtain human sequences with homology toMTSP protein) by allowing for greater or lesser degrees of nucleotidesequence similarity between the known nucleotide sequence and thenucleic acid homolog being isolated. For cross species hybridization,low stringency conditions are preferred. For same species hybridization,moderately stringent conditions are preferred. After successfulamplification of the nucleic acid containing all or a portion of theidentified MTSP protein sequence or of a nucleic acid encoding all or aportion of an MTSP protein homolog, that segment may be molecularlycloned and sequenced, and used as a probe to isolate a complete cDNA orgenomic clone. This, in turn, will permit the determination of thegene's complete nucleotide sequence, the analysis of its expression, andthe production of its protein product for functional analysis. Once thenucleotide sequence is determined, an open reading frame encoding theMTSP protein gene protein product can be determined by any method wellknown in the art for determining open reading frames, for example, usingpublicly available computer programs for nucleotide sequence analysis.Once an open reading frame is defined, it is routine to determine theamino acid sequence of the protein encoded by the open reading frame. Inthis way, the nucleotide sequences of the entire MTSP protein genes aswell as the amino acid sequences of MTSP protein proteins and analogsmay be identified.

Any eukaryotic cell potentially can serve as the nucleic acid source forthe molecular cloning of the MTSP protein gene. The nucleic acids can beisolated from vertebrate, mammalian, human, porcine, bovine, feline,avian, equine, canine, as well as additional primate sources, insects,plants, etc. The DNA may be obtained by standard procedures known in theart from cloned DNA (e.g., a DNA “library”), by chemical synthesis, bycDNA cloning, or by the cloning of genomic DNA, or fragments thereof,purified from the desired cell (see, for example, Sambrook et al., 1989,Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y.; Glover, D. M. (ed.), 1985,DNA Cloning: A Practical Approach, MRL Press, Ltd., Oxford, U.K. Vol. I,II). Clones derived from genomic DNA may contain regulatory and intronDNA regions in addition to coding regions; clones derived from cDNA willcontain only exon sequences. Whatever the source, the gene should bemolecularly cloned into a suitable vector for propagation of the gene.

In the molecular cloning of the gene from genomic DNA, DNA fragments aregenerated, some of which will encode the desired gene. The DNA may becleaved at specific sites using various restriction enzymes.Alternatively, one may use DNAse in the presence of manganese tofragment the DNA, or the DNA can be physically sheared, for example, bysonication. The linear DNA fragments can then be separated according tosize by standard techniques, including but not limited to, agarose andpolyacrylamide gel electrophoresis and column chromatography.

Once the DNA fragments are generated, identification of the specific DNAfragment containing the desired gene may be accomplished in a number ofways. For example, a portion of the MTSP protein (of any species) gene(e.g., a PCR amplification product obtained as described above or anoligonucleotide having a sequence of a portion of the known nucleotidesequence) or its specific RNA, or a fragment thereof be purified andlabeled, and the generated DNA fragments may be screened by nucleic acidhybridization to the labeled probe (Benton and Davis, Science 196:180(1977); Grunstein and Hogness, Proc. Natl. Acad. Sci. U.S.A. 72:3961(1975)). Those DNA fragments with substantial homology to the probe willhybridize. It is also possible to identify the appropriate fragment byrestriction enzyme digestion(s) and comparison of fragment sizes withthose expected according to a known restriction map if such is availableor by DNA sequence analysis and comparison to the known nucleotidesequence of MTSP protein. Further selection can be carried out on thebasis of the properties of the gene. Alternatively, the presence of thegene may be detected by assays based on the physical, chemical, orimmunological properties of its expressed product. For example, cDNAclones, or DNA clones which hybrid-select the proper mRNA, can beselected which produce a protein that, e.g., has similar or identicalelectrophoretic migration, isolectric focusing behavior, proteolyticdigestion maps, antigenic properties, serine protease activity. If ananti-MTSP protein antibody is available, the protein may be identifiedby binding of labeled antibody to the putatively MTSP proteinsynthesizing clones, in an ELISA (enzyme-linked immunosorbentassay)-type procedure.

Alternatives to isolating the MTSP protein genomic DNA include, but arenot limited to, chemically synthesizing the gene sequence from a knownsequence or making cDNA to the mRNA that encodes the MTSP protein. Forexample, RNA for cDNA cloning of the MTSP protein gene can be isolatedfrom cells expressing the protein. The identified and isolated nucleicacids can then be inserted into an appropriate cloning vector. A largenumber of vector-host systems known in the art may be used. Possiblevectors include, but are not limited to, plasmids or modified viruses,but the vector system must be compatible with the host cell used. Suchvectors include, but are not limited to, bacteriophages such as lambdaderivatives, or plasmids such as pBR322 or pUC plasmid derivatives orthe Bluescript vector (Stratagene, La Jolla, Calif.). The insertion intoa cloning vector can, for example, be accomplished by ligating the DNAfragment into a cloning vector which has complementary cohesive termini.I the complementary restriction sites used to fragment the DNA are notpresent in the cloning vector, the ends of the DNA molecules may beenzymatically modified. Alternatively, any site desired may be producedby ligating nucleotide sequences (linkers) onto the DNA termini; theseligated linkers may comprise specific chemically synthesizedoligonucleotides encoding restriction endonuclease recognitionsequences. In an alternative method, the cleaved vector and MTSP proteingene may be modified by homopolymeric tailing. Recombinant molecules canbe introduced into host cells via transformation, transfection,infection, electroporation, etc., so that many copies of the genesequence are generated.

In an alternative method, the desired gene may be identified andisolated after insertion into a suitable cloning vector in a “shot gun”approach. Enrichment for the desired gene, for example, by sizefractionization, can be done before insertion into the cloning vector.

In specific embodiments, transformation of host cells with recombinantDNA molecules that incorporate the isolated MTSP protein gene, cDNA, orsynthesized DNA sequence enables generation of multiple copies of thegene. Thus, the gene may be obtained in large quantities by growingtransformants, isolating the recombinant

DNA molecules from the transformants and, when necessary, retrieving theinserted gene from the isolated recombinant DNA.

E. VECTORS, PLASMIDS AND CELLS THAT CONTAIN NUCLEIC ACIDS ENCODING ANMTSP PROTEIN OR PROTEASE DOMAIN THEREOF AND EXPRESSION OF MTSP PROTEINSVECTORS AND CELLS

For recombinant expression of one or more of the MTSP proteins, thenucleic acid containing all or a portion of the nucleotide sequenceencoding the MTSP protein can be inserted into an appropriate expressionvector, i.e., a vector that contains the necessary elements for thetranscription and translation of the inserted protein coding sequence.The necessary transcriptional and translational signals can also besupplied by the native promoter for MTSP genes, and/or their flankingregions.

Also provided are vectors that contain nucleic acid encoding the MTSPs.Cells containing the vectors are also provided. The cells includeeukaryotic and prokaryotic cells, and the vectors are any suitable foruse therein.

Prokaryotic and eukaryotic cells, including endothelial cells,containing the vectors are provided. Such cells include bacterial cells,yeast cells, fungal cells plant cells, insect cells and animal cells.The cells are used to produce an MTSP protein or protease domain thereofby growing the above-described cells under conditions whereby theencoded MTSP protein or protease domain of the MTSP protein is expressedby the cell, and recovering the expressed protease domain protein. Forpurposes herein, the protease domain is preferably secreted into themedium.

In one embodiment, the vectors include a sequence of nucleotides thatencodes a polypeptide that has protease activity and contains all or aportion of only the protease domain, or multiple copies thereof, of anMTSP protein are provided. Also provided are vectors that comprise asequence of nucleotides that encodes the protease domain and additionalportions of an MTSP protein up to and including a full length MTSPprotein, as well as multiple copies thereof, are also provided. Thevectors may selected for expression of the MTSP protein or proteasedomain thereof in the cell or such that the MTSP protein is expressed asa transmembrane protein. Alternatively, the vectors may include signalsnecessary for secretion of encoded proteins. When the protease domain isexpressed the nucleic acid is preferably linked to a secretion signal,such as the Saccharomyces cerevisiae a mating factor signal sequence ora portion thereof.

A variety of host-vector systems may be used to express the proteincoding sequence. These include but are not limited to mammalian cellsystems infected with virus (e.g. vaccinia virus, adenovirus, etc.);insect cell systems infected with virus (e.g. baculovirus);microorganisms such as yeast containing yeast vectors; or bacteriatransformed with bacteriophage, DNA, plasmid DNA, or cosmid DNA. Theexpression elements of vectors vary in their strengths andspecificities. Depending on the host-vector system used, any one of anumber of suitable transcription and translation elements may be used.

Any methods known to those of skill in the art for the insertion of DNAfragments into a vector may be used to construct expression vectorscontaining a chimeric gene containing appropriatetranscriptional/translational control signals and protein codingsequences. These methods may include in vitro recombinant DNA andsynthetic techniques and in vivo recombinants (genetic recombination).Expression of nucleic acid sequences encoding MTSP protein, or domains,derivatives, fragments or homologs thereof, may be regulated by a secondnucleic acid sequence so that the genes or fragments thereof areexpressed in a host transformed with the recombinant DNA molecule(s).For example, expression of the proteins may be controlled by anypromoter/enhancer known in the art. In a specific embodiment, thepromoter is not native to the genes for MTSP protein. Promoters whichmay be used include but are not limited to the SV40 early promoter(Bernoist and Chambon, Nature 290:304-310 (1981)), the promotercontained in the 3′ long terminal repeat of Rous sarcoma virus (Yamamotoet al., Cell 22:787-797 (1980)), the herpes thymidine kinase promoter(Wagner et al., Proc. Natl. Acad. Sci USA 78:1441-1445 (1981)), theregulatory sequences of the metallothionein gene (Brinster et al.,Nature 296:39-42 (1982)); prokaryotic expression vectors such as theβ-lactamase promoter (Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA75:3727-3731 1978)) or the tac promoter (DeBoer et al., Proc. Natl.Acad. Sci. USA 80:21-25 (1983)); see also “Useful Proteins fromRecombinant Bacteria”: in Scientific American 242:79-94 (1980)); plantexpression vectors containing the nopaline synthetase promoter(Herrar-Estrella et al., Nature 303:209-213 (1984)) or the cauliflowermosaic virus 35S RNA promoter (Garder et al., Nucleic Acids Res. 9:2871(1981)), and the promoter of the photosynthetic enzyme ribulosebisphosphate carboxylase (Herrera-Estrella et al., Nature 310:115-120(1984)); promoter elements from yeast and other fungi such as the Gal4promoter, the alcohol dehydrogenase promoter, the phosphoglycerol kinasepromoter, the alkaline phosphatase promoter, and the following animaltranscriptional control regions that exhibit tissue specificity and havebeen used in transgenic animals: elastase I gene control region which isactive in pancreatic acinar cells (Swift et al., Cell 38:639-646 (1984);Ornitz et al., Cold Spring Harbor Symp. Quant. Biol. 50:399-409 (1986);MacDonald, Hepatology 7:425-515 (1987)); insulin gene control regionwhich is active in pancreatic beta cells (Hanahan et al., Nature315:115-122 (1985)), immunoglobulin gene control region which is activein lymphoid cells (Grosschedl et al., Cell 38:647-658 (1984); Adams etal., Nature 318:533-538 (1985); Alexander et al., Mol. Cell Biol.7:1436-1444 (1987)), mouse mammary tumor virus control region which isactive in testicular, breast, lymphoid and mast cells (Leder et al.,Cell 45:485-495 (1986)), albumin gene control region which is active inliver (Pinckert et al., Genes and Devel. 1:268-276 (1987)),alpha-fetoprotein gene control region which is active in liver (Krumlaufet al., Mol. Cell. Biol. 5:1639-1648 (1985); Hammer et al., Science235:53-58 1987)), alpha-1 antitrypsin gene control region which isactive in liver (Kelsey et al., Genes and Devel. 1:161-171 (1987)), betaglobin gene control region which is active in myeloid cells (Mogram etal., Nature 315:338-340 (1985); Kollias et al., Cell 46:89-94 (1986)),myelin basic protein gene control region which is active inoligodendrocyte cells of the brain (Readhead et al., Cell 48:703-712(1987)), myosin light chain-2 gene control region which is active inskeletal muscle (Sani, Nature 314:283-286 (1985)), and gonadotrophicreleasing hormone gene control region which is active in gonadotrophs ofthe hypothalamus (Mason et al., Science 234:1372-1378 (1986)).

In a specific embodiment, a vector is used that contains a promoteroperably linked to nucleic acids encoding an MTSP protein, or a domain,fragment, derivative or homolog, thereof, one or more origins ofreplication, and optionally, one or more selectable markers (e.g., anantibiotic resistance gene). Expression vectors containing the codingsequences, or portions thereof, of an MTSP protein, is made, forexample, by subcloning the coding portions into the EcoRI restrictionsite of each of the three pGEX vectors (glutathione S-transferaseexpression vectors (Smith and Johnson, Gene 7:31-40 (1988)). This allowsfor the expression of products in the correct reading frame. Preferredvectors and systems for expression of the protease domains of the MTSPproteins are well known Pichia vectors (available, for example, fromInvitrogen, San Diego, Calif.), particularly those designed forsecretion of the encoded proteins. One exemplary vector is described inthe EXAMPLES.

Plasmids for transformation of E. coli cells, include, for example, thepET expression vectors (see, U.S. Pat. No. 4,952,496; available fromNOVAGEN, Madison, Wis.; see, also literature published by Novagendescribing the system). Such plasmids include pET 11a, which containsthe T7lac promoter, T7 terminator, the inducible E. coli lac operator,and the lac repressor gene; pET 12a-c, which contains the T7 promoter,T7 terminator, and the E. coli ompT secretion signal; and pET 15b andpET19b (NOVAGEN, Madison, Wis.), which contain a His-TagTM leadersequence for use in purification with a His column and a thrombincleavage site that permits cleavage following purification over thecolumn; the T7-lac promoter region and the T7 terminator.

The vectors are introduced into host cells, such as Pichia cells andbacterial cells, such as E. coli, and the proteins expressed therein.Preferred Pichia strains, include, for example, GS115. Preferredbacterial hosts contain chromosomal copies of DNA encoding T7 RNApolymerase operably linked to an inducible promoter, such as the lacUVpromoter (see, U.S. Pat. No. 4,952,496). Such hosts include, but are notlimited to, the lysogenic E. coli strain BL21(DE3).

Expression and Production of Proteins

The MTSP domains, derivatives and analogs be produced by various methodsknown in the art. For example, once a recombinant cell expressing anMTSP protein, or a domain, fragment or derivative thereof, isidentified, the individual gene product can be isolated and analyzed.This is achieved by assays based on the physical and/or functionalproperties of the protein, including, but not limited to, radioactivelabeling of the product followed by analysis by gel electrophoresis,immunoassay, cross-linking to marker-labeled product. The MTSP proteinproteins may be isolated and purified by standard methods known in theart (either from natural sources or recombinant host cells expressingthe complexes or proteins), including but not restricted to columnchromatography (e.g., ion exchange, affinity, gel exclusion,reversed-phase high pressure, fast protein liquid, etc.), differentialcentrifugation, differential solubility, or by any other standardtechnique used for the purification of proteins. Functional propertiesmay be evaluated using any suitable assay known in the art.

Alternatively, once an MTSP protein or its domain or derivative isidentified, the amino acid sequence of the protein can be deduced fromthe nucleotide sequence of the gene which encodes it. As a result, theprotein or its domain or derivative can be synthesized by standardchemical methods known in the art (e.g. see Hunkapiller et al, Nature310:105-111 (1984)).

Manipulations of MTSP protein sequences may be made at the proteinlevel. Also contemplated herein are MTSP protein proteins, domainsthereof, derivatives or analogs or fragments thereof, which aredifferentially modified during or after translation, e.g., byglycosylation, acetylation, phosphorylation, amidation, derivatizationby known protecting/blocking groups, proteolytic cleavage, linkage to anantibody molecule or other cellular ligand, etc. Any of numerouschemical modifications may be carried out by known techniques, includingbut not limited to specific chemical cleavage by cyanogen bromide,trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin, etc.

In addition, domains, analogs and derivatives of an MTSP protein can bechemically synthesized. For example, a peptide corresponding to aportion of an MTSP protein, which includes the desired domain or whichmediates the desired activity in vitro can be synthesized by use of apeptide synthesizer. Furthermore, if desired, nonclassical amino acidsor chemical amino acid analogs can be introduced as a substitution oraddition into the MTSP protein sequence. Non-classical amino acidsinclude but are not limited to the D-isomers of the common amino acids,a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid,∈-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid,3-amino propionoic acid, ornithine, norleucine, norvaline,hydroxyproline, sarcosine, citrulline, cysteic acid, t-butylglycine,t-butylalanine, phenylglycine, cyclohexylalanine, β-alanine,fluoro-amino acids, designer amino acids such as β-methyl amino acids,Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs ingeneral. Furthermore, the amino acid can be D (dextrorotary) or L(levorotary).

In cases where natural products are suspected of being mutant or areisolated from new species, the amino acid sequence of the MTSP proteinisolated from the natural source, as well as those expressed in vitro,or from synthesized expression vectors in vivo or in vitro, can bedetermined from analysis of the DNA sequence, or alternatively, bydirect sequencing of the isolated protein. Such analysis may beperformed by manual sequencing or through use of an automated amino acidsequenator.

Modifications

A variety of modification of the MTSP proteins and domains arecontemplated herein. An MTSP-encoding nucleic acid molecule may bemodified by any of numerous strategies known in the art (Sambrook etal., 1990, Molecular Cloning, A Laboratory Manual, 2d ed., Cold SpringHarbor Laboratory, Cold Spring Harbor, N.Y.). The sequences can becleaved at appropriate sites with restriction endonuclease(s), followedby further enzymatic modification if desired, isolated, and ligated invitro. In the production of the gene encoding a domain, derivative oranalog of MTSP, care should be taken to ensure that the modified generetains the original translational reading frame, uninterrupted bytranslational stop signals, in the gene region where the desiredactivity is encoded.

Additionally, the MTSP-encoding nucleic acid molecules can be mutated invitro or in vivo, to create and/or destroy translation, initiation,and/or termination sequences, or to create variations in coding regionsand/or form new restriction endonuclease sites or destroy pre-existingones, to facilitate further in vitro modification. Also, as describedherein muteins with primary sequence alterations, such as replacementsof Cys residues and elimination of glycosylation sites are contemplated.Such mutations may be effected by any technique for mutagenesis known inthe art, including, but not limited to, chemical mutagenesis and invitro site-directed mutagenesis (Hutchinson et al., J. Biol. Chem.253:6551-6558 (1978)), use of TAB® linkers (Pharmacia). In oneembodiment, for example, an MTSP protein or domain thereof is modifiedto include a fluorescent label. In other specific embodiments, the MTSPprotein is modified to have a heterofunctional reagent, suchheterofunctional reagents can be used to crosslink the members of thecomplex. The MTSP proteins may be isolated and purified by standardmethods known in the art (either from natural sources or recombinanthost cells expressing the complexes or proteins), including but notrestricted to column chromatography (e.g., ion exchange, affinity, gelexclusion, reversed-phase high pressure, fast protein liquid, etc.),differential centrifugation, differential solubility, or by any otherstandard technique used for the purification of proteins. Functionalproperties may be evaluated using any suitable assay known in the art.

Alternatively, once a MTSP or its domain or derivative is identified,the amino acid sequence of the protein can be deduced from thenucleotide sequence of the gene which encodes it. As a result, theprotein or its domain or derivative can be synthesized by standardchemical methods known in the art (e.g., see Hunkapiller et al, Nature,310:105-111 (1984)).

Manipulations of MTSP sequences may be made at the protein level. MTSPdomains, derivatives or analogs or fragments, which are differentiallymodified during or after translation, e.g., by glycosylation,acetylation, phosphorylation, amidation, derivatization by knownprotecting/blocking groups, proteolytic cleavage, linkage to an antibodymolecule and other cellular ligand, are contemplated herein. Any ofnumerous chemical modifications may be carried out by known techniques,including but not limited to specific chemical cleavage by cyanogenbromide, trypsin, chymotrypsin, papain, V8 protease, NaBH₄, acetylation,formylation, oxidation, reduction, metabolic synthesis in the presenceof tunicamycin, etc.

In addition, domains, analogs and derivatives of a MTSP can bechemically synthesized. For example, a peptide corresponding to aportion of a MTSP, which comprises the desired domain or which mediatesthe desired activity in vitro can be synthesized by use of a peptidesynthesizer. Furthermore, if desired, nonclassical amino acids orchemical amino acid analogs can be introduced as a substitution oraddition into the MTSP sequence. Non-classical amino acids include butare not limited to the D-isomers of the common amino acids, a-aminoisobutyric acid, 4-aminobutyric acid, Abu, 2-aminobutyric acid, ∈-Abu,e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-aminopropionoic acid, ornithine, norleucine, norvaline, hydroxyproline,sarcosine, citrulline, cysteic acid, t-butylglycine, t-butylalanine,phenylglycine, cyclohexylalanine, β-alanine, fluoro-amino acids,designer amino acids such as β-methyl amino acids, Ca-methyl aminoacids, Na-methyl amino acids, and amino acid analogs in general.Furthermore, the amino acid can be D (dextrorotary) or L (levorotary).

F. SCREENING METHODS

The single chain protease domains, as shown herein, can be used in avariety of methods to identify compounds that modulate the activitythereof For MTSPs that exhibit higher activity or expression in tumorcells, compounds that inhibit the proteolytic activity are of particularinterest. For any MTSPs that are active at lower levels in tumor cells,compounds or agents that enhance the activity are potentially ofinterest. In all instances the identified compounds will include agentsthat are candidate cancer treatments.

Several types of assays are exemplified and described herein. It isunderstood that the protease domains may be used in other assays. It isshown here, however, that the single chain protease domains exhibitcatalytic activity. As such they are ideal for in vitro screeningassays.

They may also be used in binding assays.

The MTSP3, MTSP4 and MTSP6 full length zymogens, activated enzymes,single and double chain protease domains are contemplated for use in anyscreening assay known to those of skill in the art, including thoseprovided herein. Hence the following description, if directed toproteolytic assays is intended to apply to use of a single chainprotease domain or a catalytically active portion thereof of any MTSP,including an MTSP3, MTSP4 or an MTSP6. Other assays, such as bindingassays are provided herein, particularly for use with an MTSP3, MTSP4 orMTSP6, including any variants, such as splice variants thereof. MTSP3and MTSP4 are of most interest in such assays.

1. Catalytic Assays for Identification of Agents that Modulate theProtease Activity of an MTSP Protein

Methods for identifying a modulator of the catalytic activity of anMTSP, particularly a single chain protease domain or catalyticallyactive portion thereof, are provided herein. The methods can bepracticed by: a) contacting the MTSP, particularly a single-chain domainthereof, with a substrate of the MTSP in the presence of a testsubstance, and detecting the proteolysis of the substrate, whereby theactivity of the MTSP is assessed, and comparing the activity to acontrol. For example, the control can be the activity of the MTSPassessed by contacting an MTSP, particularly a single-chain domainthereof, with a substrate of the MTSP, and detecting the proteolysis ofthe substrate, whereby the activity of the MTSP is assessed. The resultsin the presence and absence of the test compounds are compared. Adifference in the activity indicates that the test substance modulatesthe activity of the MTSP.

In one embodiment a plurality of the test substances are screenedsimultaneously in the above screening method. In another embodiment, theMTSP is isolated from a target cell as a means for then identifyingagents that are potentially specific for the target cell.

In still another embodiment, The test substance is a therapeuticcompound, and whereby a difference of the MTSP activity measured in thepresence and in the absence of the test substance indicates that thetarget cell responds to the therapeutic compound.

One method include the steps of (a) contacting the MTSP protein orprotease domain thereof with one or a plurality of test compounds underconditions conducive to interaction between the ligand and thecompounds; and (b) identifying one or more compounds in the pluralitythat specifically binds to the ligand.

Another method provided herein includes the steps of a) contacting anMTSP protein or protease domain thereof with a substrate of the MTSPprotein, and detecting the proteolysis of the substrate, whereby theactivity of the MTSP protein is assessed; b) contacting the MTSP proteinwith a substrate of the MTSP protein in the presence of a testsubstance, and detecting the proteolysis of the substrate, whereby theactivity of the MTSP protein is assessed; and c) comparing the activityof the MTSP protein assessed in steps a) and b), whereby the activitymeasured in step a) differs from the activity measured in step b)indicates that the test substance modulates the activity of the MTSPprotein.

In another embodiment, a plurality of the test substances are screenedsimultaneously. In comparing the activity of an MTSP protein in thepresence and absence of a test substance to assess whether the testsubstance is a modulator of the MTSP protein, it is unnecessary to assaythe activity in parallel, although such parallel measurement ispreferred. It is possible to measure the activity of the MTSP protein atone time point and compare the measured activity to a historical valueof the activity of the MTSP protein.

For instance, one can measure the activity of the MTSP protein in thepresence of a test substance and compare with historical value of theactivity of the MTSP protein measured previously in the absence of thetest substance, and vice versa. This can be accomplished, for example,by providing the activity of the MTSP protein on an insert or pamphletprovided with a kit for conducting the assay.

Methods for selecting substrates for a particular MTSP are described inthe EXAMPLES, and particular proteolytic assays are exemplified.

Combinations and kits containing the combinations optionally includinginstructions for performing the assays are provided. The combinationsinclude an MTSP protein and a substrate of the MTSP protein to beassayed; and, optionally reagents for detecting proteolysis of thesubstrate. The substrates, which are typically proteins subject toproteolysis by a particular MTSP protein, can be identified empiricallyby testing the ability of the MTSP protein to cleave the test substrate.Substrates that are cleaved most effectively (i.e., at the lowestconcentrations and/or fastest rate or under desirable conditions), areidentified.

Additionally provided herein is a kit containing the above-describedcombination. Preferably, the kit further includes instructions foridentifying a modulator of the activity of an MTSP protein. Any MTSPprotein is contemplated as target for identifying modulators of theactivity thereof

2. Binding Assays

Also provided herein are methods for identification and isolation ofagents, particularly compounds that bind to MTSPs. The assays aredesigned to identify agents that bind to the zymogen form, the singlechain isolated protease domain (or a protein, other than an MTSPprotein, that contains the protease domain of an MTSP protein), and tothe activated form, including the activated form derived from the fulllength zymogen or from an extended protease domain. The identifiedcompounds are candidates or leads for identification of compounds fortreatments of tumors and other disorders and diseases involving aberrantangiogenesis. The MTSP proteins used in the methods include any MTSPprotein as defined herein, and preferably use MTSP single chain domainor proteolytically active portion thereof.

A variety of methods are provided herein. These methods may be performedin solution or in solid phase reactions in which the MTSP protein(s) orprotease domain(s) thereof are linked, either directly or indirectly viaa linker, to a solid support. Screening assays are described in theExamples, and these assays have been used to identify candidatecompounds.

For purposes herein, all binding assays described above are provided forMTSP3, MTSP4 and MTSP6. For MTSP1 (including any variant thereof) andother such proteases, binding assays that employ the isolated singlechain protease domain or a protein containing such domain (other thanthe MTSP from which the protease is derived) are provided.

Methods for identifying an agent, such as a compound, that specificallybinds to an MTSP single chain protease domain or an MTSP, such as anMTSP3, MTSP4 or an MTSP6, are provided herein. The method can bepracticed by (a) contacting the MTSP with one or a plurality of testagents under conditions conducive to binding between the MTSP and anagent; and (b) identifying one or more agents within the plurality thatspecifically binds to the MTSP.

For example, in practicing such methods the MTSP polypeptide is mixedwith a potential binding partner or an extract or fraction of a cellunder conditions that allow the association of potential bindingpartners with the polypeptide. After mixing, peptides, polypeptides,proteins or other molecules that have become associated with an MTSP areseparated from the mixture. The binding partner that bound to the MTSPcan then be removed and further analyzed. To identify and isolate abinding partner, the entire protein, for instance the entire disclosedprotein of SEQ ID Nos. 6, 8 10 or 12 can be used. Alternatively, afragment of the protein can be used.

A variety of methods can be used to obtain cell extracts. Cells can bedisrupted using either physical or chemical disruption methods. Examplesof physical disruption methods include, but are not limited to,sonication and mechanical shearing. Examples of chemical lysis methodsinclude, but are not limited to, detergent lysis and enzyme lysis. Askilled artisan can readily adapt methods for preparing cellularextracts in order to obtain extracts for use in the present methods.

Once an extract of a cell is prepared, the extract is mixed with theMTSP under conditions in which association of the protein with thebinding partner can occur. A variety of conditions can be used, the mostpreferred being conditions that closely resemble conditions found in thecytoplasm of a human cell. Features such as osmolarity, pH, temperature,and the concentration of cellular extract used, can be varied tooptimize the association of the protein with the binding partner.

After mixing under appropriate conditions, the bound complex isseparated from the mixture. A variety of techniques can be used toseparate the mixture. For example, antibodies specific to an MTSP can beused to immunoprecipitate the binding partner complex. Alternatively,standard chemical separation techniques such as chromatography anddensity/sediment centrifugation can be used.

After removing the non-associated cellular constituents in the extract,the binding partner can be dissociated from the complex usingconventional methods. For example, dissociation can be accomplished byaltering the salt concentration or pH of the mixture.

To aid in separating associated binding partner pairs from the mixedextract, the MTSP can be immobilized on a solid support. For example,the protein can be attached to a nitrocellulose matrix or acrylic beads.Attachment of the protein or a fragment thereof to a solid support aidsin separating peptide/binding partner pairs from other constituentsfound in the extract. The identified binding partners can be either asingle protein or a complex made up of two or more proteins.

Alternatively, the nucleic acid molecules encoding the single chainproteases can be used in a yeast two-hybrid system. The yeast two-hybridsystem has been used to identify other protein partner pairs and canreadily be adapted to employ the nucleic acid molecules hereindescribed.

Another in vitro binding assay, particularly for an MTSP3, MTSP4 or anMTSP6 uses a mixture of a polypeptide that contains at least thecatalytic domain of one of these proteins and one or more candidatebinding targets or substrates. After incubating the mixture underappropriate conditions, one determines whether the MTSP or a polypeptidefragment thereof containing the catalytic domain binds with thecandidate substrate. For cell-free binding assays, one of the componentsincludes or is coupled to a detectable label. The label may provide fordirect detection, such as radioactivity, luminescence, optical orelectron density, etc., or indirect detection such as an epitope tag, anenzyme, etc. A variety of methods may be employed to detect the labeldepending on the nature of the label and other assay components. Forexample, the label may be detected bound to the solid substrate or aportion of the bound complex containing the label may be separated fromthe solid substrate, and the label thereafter detected.

3. Detection of Signal Transduction

The cell surface location of the MTSPs suggests a role for some or allof these proteins in signal transduction. Assays for assessing signaltransduction are well known to those of skill in the art, and may beadapted for use with the MTSP protein.

Assays for identifying agents that effect or alter signal transductionmediated by an MTSP, particularly the full length or a sufficientportion to anchor the extracellular domain or a function portion thereofof an MTSP on the surface of a cell are provided. Such assays, include,for example, transcription based assays in which modulation of atransduced signal is assessed by detecting an effect on an expressionfrom a reporter gene (see, e.g., U.S. Pat. No. 5,436,128).

4. Methods for Identifying Agents that Modulate the Expression a NucleicAcid Encoding an MTSP, Particularly an MTSP3, MTSP4 or MTSP6

Another embodiment provides methods for identifying agents that modulatethe expression of a nucleic acid encoding an MTSP, particularly anMTSP3, MTSP4 or MTSP. Such assays use any available means of monitoringfor changes in the expression level of the nucleic acids encoding anMTSP, such as MTSP3 or MTSP4.

In one assay format, cell lines that contain reporter gene fusionsbetween the open reading frame of MTSP3, MTSP4 or MTSP6 or a domainthereof, particularly the protease domain and any assayable fusionpartner may be prepared. Numerous assayable fusion partners are knownand readily available including the firefly luciferase gene and the geneencoding chloramphenicol acetyltransferase (Alam et al., Anal. Biochem.188: 245-54 (1990)). Cell lines containing the reporter gene fusions arethen exposed to the agent to be tested under appropriate conditions andtime. Differential expression of the reporter gene between samplesexposed to the agent and control samples identifies agents whichmodulate the expression of a nucleic acid encoding an MTSP3, MTSP4 orMTSP6.

Additional assay formats may be used to monitor the ability of the agentto modulate the expression of a nucleic acid encoding an MTSP3, MTSP4 orMTSP6. For instance, mRNA expression may be monitored directly byhybridization to the nucleic acids. Cell lines are exposed to the agentto be tested under appropriate conditions and time and total RNA or mRNAis isolated by standard procedures (see, e.g., Sambrook et al. (1989)MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed. Cold Spring HarborLaboratory Press). Probes to detect differences in RNA expression levelsbetween cells exposed to the agent and control cells may be preparedfrom the nucleic acids. It is preferable, but not necessary, to designprobes which hybridize only with target nucleic acids under conditionsof high stringency. Only highly complementary nucleic acid hybrids formunder conditions of high stringency. Accordingly, the stringency of theassay conditions determines the amount of complementarity which shouldexist between two nucleic acid strands in order to form a hybrid.Stringency should be chosen to maximize the difference in stabilitybetween the probe:target hybrid and potential probe:non-target hybrids.

Probes may be designed from the nucleic acids through methods known inthe art. For instance, the G+C content of the probe and the probe lengthcan affect probe binding to its target sequence. Methods to optimizeprobe specificity are commonly available (see, e.g., Sambrook et al.(1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed. Cold SpringHarbor Laboratory Press); and Ausubel et al. (1995) CURRENT PROTOCOLS INMOLECULAR BIOLOGY, Greene Publishing Co., NY).

Hybridization conditions are modified using known methods (see, e.g.,Sambrook et al. (1989) MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed.Cold Spring Harbor Laboratory Press); and Ausubel et al. (1995) CURRENTPROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Co., NY), as requiredfor each probe. Hybridization of total cellular RNA or RNA enriched forpolyA RNA can be accomplished in any available format. For instance,total cellular RNA or RNA enriched for polyA RNA can be affixed to asolid support, and the solid support exposed to at least one probecomprising at least one, or part of one of the nucleic acid moleculesunder conditions in which the probe will specifically hybridize.Alternatively, nucleic acid fragments comprising at least one, or partof one of the sequences can be affixed to a solid support, such as aporous glass wafer. The glass wafer can then be exposed to totalcellular RNA or polyA RNA from a sample under conditions in which theaffixed sequences will specifically hybridize. Such glass wafers andhybridization methods are widely available, for example, those disclosedby Beattie (WO 95/11755). By examining for the ability of a given probeto specifically hybridize to an RNA sample from an untreated cellpopulation and from a cell population exposed to the agent, agents whichup or down regulate the expression of a nucleic acid encoding theprotein having the sequence of SEQ ID NO:3 or SEQ ID NO:4 areidentified.

5. Methods for Identifying Agents that Modulate at Least One Activity ofan MTPS, Such as MTSP3, MTSP4 or MTSP6

Methods for identifying agents that modulate at least one activity of aan MTSP, such as an MTSP3, MTSP4 or MTSP6 are provided. Such methods orassays may use any means of monitoring or detecting the desiredactivity.

In one format, the relative amounts of a protein between a cellpopulation that has been exposed to the agent to be tested compared toan un-exposed control cell population may be assayed (e.g., a prostatecancer cell line, a lung cancer cell line, a colon cancer cell line or abreast cancer cell line). In this format, probes, such as specificantibodies, are used to monitor the differential expression of theprotein in the different cell populations. Cell lines or populations areexposed to the agent to be tested under appropriate conditions and time.Cellular lysates may be prepared from the exposed cell line orpopulation and a control, unexposed cell line or population. Thecellular lysates are then analyzed with the probe.

For example, N- and C-terminal fragments of the MTSP can be expressed inbacteria and used to search for proteins which bind to these fragments.Fusion proteins, such as His-tag or GST fusion to the N- or C-terminalregions of the MTSP, such as an MTSP3, MTSP4 or an MTSP6, can beprepared for use as a substrate. These fusion proteins can be coupledto, for example, Glutathione-Sepharose beads and then probed with celllysates. Prior to lysis, the cells may be treated with a candidate agentwhich may modulate an MTSP, such as an MTSP3, MTSP4 or an MTSP6, orproteins that interact with domains thereon. Lysate proteins binding tothe fusion proteins can be resolved by SDS-PAGE, isolated and identifiedby protein sequencing or mass spectroscopy, as is known in the art.

Antibody probes are prepared by immunizing suitable mammalian hosts inappropriate immunization protocols using the peptides, polypeptides orproteins if they are of sufficient length (e.g., 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or more consecutive amino acidsthe MTSP protein, such as an MTSP3, an MTSP4 or an MTSP6), or ifrequired to enhance immunogenicity, conjugated to suitable carriers.Methods for preparing immunogenic conjugates with carriers, such asbovine serum albumin (BSA), keyhole limpet hemocyanin (KLH), or othercarrier proteins are well known in the art. In some circumstances,direct conjugation using, for example, carbodiimide reagents may beeffective; in other instances linking reagents such as those supplied byPierce Chemical Co., Rockford, Ill., may be desirable to provideaccessibility to the hapten. Hapten peptides can be extended at eitherthe amino or carboxy terminus with a Cys residue or interspersed withcysteine residues, for example, to facilitate linking to a carrier.Administration of the immunogens is conducted generally by injectionover a suitable time period and with use of suitable adjuvants, as isgenerally understood in the art. During the immunization schedule,titers of antibodies are taken to determine adequacy of antibodyformation.

Anti-peptide antibodies can be generated using synthetic peptidescorresponding to, for example, the carboxy terminal amino acids of theMTSP. Synthetic peptides can be as small as 1-3 amino acids in length,but are preferably at least 4 or more amino acid residues long. Thepeptides can be coupled to KLH using standard methods and can beimmunized into animals, such as rabbits or ungulate. Polyclonalantibodies can then be purified, for example using Actigel beadscontaining the covalently bound peptide.

While the polyclonal antisera produced in this way may be satisfactoryfor some applications, for pharmaceutical compositions, use ofmonoclonal preparations is preferred. Immortalized cell lines whichsecrete the desired monoclonal antibodies may be prepared using thestandard method of Kohler et al., (Nature 256:495-7 (1975)) ormodifications which effect immortalization of lymphocytes or spleencells, as is generally known. The immortalized cell lines secreting thedesired antibodies are screened by immunoassay in which the antigen isthe peptide hapten, polypeptide or protein. When the appropriateimmortalized cell culture secreting the desired antibody is identified,the cells can be cultured either in vitro or by production in vivo viaascites fluid. Of particular interest, are monoclonal antibodies thatrecognize the catalytic domain of an MTSP, such as an MTSP3, MTSP4 or anMTSP6.

Additionally, the zymogen or two-chain forms the MTSP can be used tomake monoclonal antibodies which recognize conformation epitopes. Forpeptide-directed monoclonal antibodies, peptides from the C1r/C1s domaincan be used to generate anti-C1r/C1s domain monoclonal antibodies whichcan thereby block activation of the zymogen to the two-chain form of theMTSP. This domain can similarly be the substrate for other non-antibodycompounds which bind to these preferred domains on either thesingle-chain or double-chain forms of the MTSP3, MTSP4 or MTSP6, andthereby modulate the activity of thereof or prevent its activation.

The desired monoclonal antibodies are then recovered from the culturesupernatant or from the ascites supernatant. Fragments of themonoclonals or the polyclonal antisera which contain the immunologicallysignificant portion can be used as antagonists, as well as the intactantibodies. Use of immunologically reactive fragments, such as the Fab,Fab′, of F(ab′)2 fragments are often preferable, especially in atherapeutic context, as these fragments are generally less immunogenicthan the whole immunoglobulin.

The antibodies or fragments may also be produced. Regions that bindspecifically to the desired regions of receptor can also be produced inthe context of chimeras with multiple species origin.

Agents that are assayed in the above method can be randomly selected orrationally selected or designed.

The agents can be, as examples, peptides, small molecules, andcarbohydrates. A skilled artisan can readily recognize that there is nolimit as to the structural nature of the agents.

The peptide agents can be prepared using standard solid phase (orsolution phase) peptide synthesis methods, as is known in the art. Inaddition, the DNA encoding these peptides may be synthesized usingcommercially available oligonucleotide synthesis instrumentation andproduced recombinantly using standard recombinant production systems.The production using solid phase peptide synthesis is necessitated ifnon-gene-encoded amino acids are to be included.

G. ASSAY FORMATS AND SELECTION OF TEST SUBSTANCES

A variety of formats and detection protocols are known for performingscreening assays. Any such formats and protocols may be adapted foridentifying modulators of MTSP protein activities. The followingincludes a discussion of exemplary protocols.

1. High Throughput Screening Assays

Although the above-described assay can be conducted where a single MTSPprotein is screened, and/or a single test substance is screened for inone assay, the assay is preferably conducted in a high throughputscreening mode, i.e., a plurality of the MTSP proteins are screenedagainst and/or a plurality of the test substances are screened forsimultaneously (See generally, High Throughput Screening: The Discoveryof Bioactive Substances (Devlin, Ed.) Marcel Dekker, 1997; Sittampalamet al., Curr. Opin. Chem. Biol., 1(3):384-91 (1997); and Silverman etal., Curr. Opin. Chem. Biol., 2(3):397-403 (1998)). For example, theassay can be conducted in a multi-well (e.g., 24-, 48-, 96-, or384-well), chip or array format.

High-throughput screening (HTS) is the process of testing a large numberof diverse chemical structures against disease targets to identify“hits” (Sittampalam et al., Curr. Opin. Chem. Biol., 1(3):384-91(1997)). Current state-of-the-art HTS operations are highly automatedand computerized to handle sample preparation, assay procedures and thesubsequent processing of large volumes of data.

Detection technologies employed in high-throughput screens depend on thetype of biochemical pathway being investigated (Sittampalam et al.,Curr. Opin. Chem. Biol., 1(3):384-91 (1997)). These methods include,radiochemical methods, such as the scintillation proximity assays (SPA),which can be adapted to a variety of enzyme assays (Lerner et al., J.Biomol. Screening, 1:135-143 (1996); Baker et al., Anal. Biochem.,239:20-24 (1996); Baum et al., Anal. Biochem., 237:129-134 (1996); andSullivan et al., J. Biomol. Screening, 2:19-23 (1997)) andprotein-protein interaction assays (Braunwalder et al., J. Biomol.Screening, 1:23-26 (1996); Sonatore et al., Anal. Biochem., 240:289-297(1996); and Chen et al., J. Biol. Chem., 271:25308-25315 (1996)), andnon-isotopic detection methods, including but are not limited to,colorimetric and luminescence detection methods, resonance energytransfer (RET) methods, time-resolved fluorescence (HTRF) methods,cell-based fluorescence assays, such as fluorescence resonance energytransfer (FRET) procedures (see, e.g., Gonzalez et al., Biophys. J.,69:1272-1280 (1995)), fluorescence polarization or anisotropy methods(see, e.g., Jameson et al., Methods Enzymol., 246:283-300 (1995);Jolley, J. Biomol. Screening, 1:33-38 (1996); Lynch et al., Anal.Biochem., 247:77-82 (1997)), fluorescence correlation spectroscopy (FCS)and other such methods.

2. Test Substances

Test compounds, including small molecules and libraries and collectionsthereof can be screened in the above-described assays and assaysdescribed below to identify compounds that modulate the activity an MTSPprotein. Rational drug design methodologies that rely on computationalchemistry may be used to screen and identify candidate compounds.

The compounds identified by the screening methods include inhibitors,including antagonists, and may be agonists Compounds for screening areany compounds and collections of compounds available, know or that canbe prepared.

a. Selection of Compounds

Compounds can be selected for their potency and selectivity ofinhibition of serine proteases, especially MTSP protein. As describedherein, and as generally known, a target serine protease and itssubstrate are combined under assay conditions permitting reaction of theprotease with its substrate. The assay is performed in the absence oftest compound, and in the presence of increasing concentrations of thetest compound. The concentration of test compound at which 50% of theserine protease activity is inhibited by the test compound is the IC₅₀value (Inhibitory Concentration) or EC₅₀ (Effective Concentration) valuefor that compound. Within a series or group of test compounds, thosehaving lower IC₅₀ or EC₅₀ values are considered more potent inhibitorsof the serine protease than those compounds having higher IC₅₀ or EC₅₀values. The IC₅₀ measurement is often used for more simplistic assays,whereas the EC₅₀ is often used for more complicated assays, such asthose employing cells.

Preferred compounds according to this aspect have an IC₅₀ value of 100nM or less as measured in an in vitro assay for inhibition of MTSPprotein activity. Especially preferred compounds have an IC₅₀ value ofless than 100 nM.

The test compounds also are evaluated for selectivity toward a serineprotease.

As described herein, and as generally known, a test compound is assayedfor its potency toward a panel of serine proteases and other enzymes andan IC₅₀ value or EC₅₀ value is determined for each test compound in eachassay system. A compound that demonstrates a low IC₅₀ value or EC₅₀value for the target enzyme, e.g., MTSP protein, and a higher IC₅₀ valueor EC₅₀ value for other enzymes within the test panel (e.g., urokinasetissue plasminogen activator, thrombin, Factor Xa), is considered to beselective toward the target enzyme. Generally, a compound is deemedselective if its IC₅₀ value or EC₅₀ value in the target enzyme assay isat least one order of magnitude less than the next smallest IC₅₀ valueor EC₅₀ value measured in the selectivity panel of enzymes.

Presently preferred compounds have an IC₅₀ value of 100 nM or less asmeasured in an in vitro assay for inhibition of urokinase activity.Especially preferred compounds have an IC₅₀ value in the in vitrourokinase inhibition assay that is at least one order of magnitudesmaller than the IC₅₀ value measured in the in vitro tPA inhibitionassay. Compounds having a selectivity ratio of IC₅₀ u-PA assay: IC₅₀MTSP protein assay of greater than 100 are especially preferred.

Compounds are also evaluated for their activity in vivo. The type ofassay chosen for evaluation of test compounds will depend on thepathological condition to be treated or prevented by use of thecompound, as well as the route of administration to be evaluated for thetest compound.

For instance, to evaluate the activity of a compound to reduce tumorgrowth through inhibition of MTSP protein, the procedures described byJankun et al., Canc. Res., 57:559-563 (1997) to evaluate PAI-1 can beemployed. Briefly, the ATCC cell lines DU145 and LnCaP are injected intoSCID mice. After tumors are established, the mice are given testcompound according to a dosing regime determined from the compound's invitro characteristics. The Jankun et al. compound was administered inwater. Tumor volume measurements are taken twice a week for about fiveweeks. A compound is deemed active if an animal to which the compoundwas administered exhibited decreased tumor volume, as compared toanimals receiving appropriate control compounds.

Another in vivo experimental model designed to evaluate the effect ofp-aminobenzamidine, a swine protease inhibitor, on reducing tumor volumeis described by Billstrom et al., Int. J. Cancer, 61:542-547 (1995).

To evaluate the ability of a compound to reduce the occurrence of, orinhibit, metastasis, the procedures described by Kobayashi et al., Int.J. Canc., 57:727-733d (1994) can be employed. Briefly, a mureinxenograft selected for high lung colonization potential in injected intoC57B1/6 mice i.v. (experimental metastasis) or s.c. into the abdominalwall (spontaneous metastasis). Various concentrations of the compound tobe tested can be admixed with the tumor cells in Matrigel prior toinjection. Daily i.p. injections of the test compound are made either ondays 1-6 or days 7-13 after tumor inoculation. The animals aresacrificed about three or four weeks after tumor inoculation, and thelung tumor colonies are counted. Evaluation of the resulting datapermits a determination as to efficacy of the test compound, optimaldosing and route of administration.

The activity of the tested compounds toward decreasing tumor volume andmetastasis can be evaluated in model described in Rabbani et al., Int.J. Cancer 63:840-845 (1995) to evaluate their inhibitor. There, Mat LyLutumor cells were injected into the flank of Copenhagen rats. The animalswere implanted with osmotic minipumps to continuously administer variousdoses of test compound for up to three weeks. The tumor mass and volumeof experimental and control animals were evaluated during theexperiment, as were metastatic growths. Evaluation of the resulting datapermits a determination as to efficacy of the test compound, optimaldosing, and route of administration. Some of these authors described arelated protocol in Xing et al., Canc. Res., 57:3585-3593 (1997).

To evaluate the inhibitory activity of a compound, a rabbit corneaneovascularization model can be employed. Avery et al., Arch.Ophthalmol., 108:1474-1475 (1990) describe anesthetizing New Zealandalbino rabbits and then making a central corneal incision and forming aradial corneal pocket. A slow release prostaglandin pellet was placed inthe pocket to induce neovascularization. Test compound was administeredi.p. for five days, at which time the animals were sacrificed. Theeffect of the test compound is evaluated by review of periodicphotographs taken of the limbus, which can be used to calculate the areaof neovascular response and, therefore, limbal neovascularization. Adecreased area of neovascularization as compared with appropriatecontrols indicates the test compound was effective at decreasing orinhibiting neovascularization.

An angiogenesis model used to evaluate the effect of a test compound inpreventing angiogenesis is described by Min et al., Canc. Res.,56:2428-2433 (1996). C57BL6 mice receive subcutaneous injections of aMatrigel mixture containing bFGF, as the angiogenesis-inducing agent,with and without the test compound. After five days, the animals aresacrificed and the Matrigel plugs, in which neovascularization can bevisualized, are photographed. An experimental animal receiving Matrigeland an effective dose of test compound will exhibit less vascularizationthan a control animal or an experimental animal receiving a less- ornon-effective does of compound.

An in vivo system designed to test compounds for their ability to limitthe spread of primary tumors is described by Crowley et al., Proc. Natl.Acad. Sci., 90:5021-5025 (1993). Nude mice are injected with tumor cells(PC3) engineered to express CAT (chloramphenicol acetyltransferase).Compounds to be tested for their ability to decrease tumor size and/ormetastases are administered to the animals, and subsequent measurementsof tumor size and/or metastatic growths are made. In addition, the levelof CAT detected in various organs provides an indication of the abilityof the test compound to inhibit metastasis; detection of less CAT intissues of a treated animal versus a control animal indicates lessCAT-expressing cells migrated to that tissue.

In vivo experimental modes designed to evaluate the inhibitory potentialof a test serine protease inhibitors, using a tumor cell line F3II, theto be highly invasive, are described by Alonso et al., Breast Canc. Res.Treat., 40:209-223 (1996). This group describes in vivo studies fortoxicity determination, tumor growth, invasiveness, spontaneousmetastasis, experimental lung metastasis, and an angiogenesis assay.

The CAM model (chick embryo chorioallantoic membrane model), firstdescribed by L. Ossowski in 1998 (J. Cell Biol., 107:2437-2445 (1988)),provides another method for evaluating the urokinase inhibitory activityof a test compound. In the CAM model, tumor cells invade through thechorioallantoic membrane containing CAM with tumor cells in the presenceof several serine protease inhibitors results in less or no invasion ofthe tumor cells through the membrane. Thus, the CAM assay is performedwith CAM and tumor cells in the presence and absence of variousconcentrations of test compound. The invasiveness of tumor cells ismeasured under such conditions to provide an indication of thecompound's inhibitory activity. A compound having inhibitory activitycorrelates with less tumor invasion.

The CAM model is also used in a standard assay of angiogenesis (i.e.,effect on formation of new blood vessels (Brooks et al., Methods inMolecular Biology, 129:257-269 (1999)). According to this model, afilter disc containing an angiogenesis inducer, such as basic fibroblastgrowth factor (bFDG) is placed onto the CAM. Diffusion of the cytokineinto the CAM induces local angiogenesis, which may be measured inseveral ways such as by counting the number of blood vessel branchpoints within the CAM directly below the filter disc. The ability ofidentified compounds to inhibit cytokine-induced angiogenesis can betested using this model.

A test compound can either be added to the filter disc that contains theangiogenesis inducer, be placed directly on the membrane or beadministered systemically. The extent of new blood vessel formation inthe presence and/or absence of test compound can be compared using thismodel. The formation of fewer new blood vessels in the presence of atest compound would be indicative of anti-angiogenesis activity.Demonstration of anti-angiogenesis activity for inhibitors of an MTSPprotein indicates a role in angiogenesis for that MTSP protein.

b. Known Serine Protease Inhibitors

Compounds for screening can be serine protease inhibitors, which can betested for their ability to inhibit the activity of an MTSP,particularly an MTSP3, MTSP4, or MTSP6.

Exemplary, but not limiting serine proteases, include the followingknown serine protease inhibitors are used in the screening assays:Serine Protease Inhibitor 3 (SPI-3) (Chen, M. C., et al., Citokine,11(11):856-862 (1999)); Aprotinin (Iijima, R., et al., J. Biochem.(Tokyo), 126(5):912-916 (1999)); Kazal-type serine proteaseinhibitor-like proteins (Niimi, T., et al., Eur. J. Biochem.,266(1):282-292 (1999)); Kunitz-type serine protease inhibitor(Ravichandran, S., et al., Acta Crystallogr. D. Biol, Crystallogr.,55(11):1814-1821 (1999)); Tissue factor pathwayinhibitor-2/Matrix-associated serine rotease inhibitor (TFPI-2/MSPI),(Liu, Y., et al., Arch. Biochem. Biophys., 370(1):112-8 (1999));Bukunin, (Yi, C. Y., et al., J. Invest. Dermatol., 113(2):182-8 (1999));Nafmostat mesilate (Ryo, R., et al., Vox Sang., 76(4):241-6 (1999));TPCK (Huang, Y., et al., Oncogene, 18(23):3431-9 (1999)); A syntheticcotton-bound serine protease inhibitor (Edwards, J. V., et al., WoundRepair Regen., 7(2):106-18 (1999)); FUT-175 (Sawada, M., et al., Stroke,30(3):644-50 (1999)); Combination of serine protease inhibitor FUT-0175and thromboxane synthetase inhibitor OKY-046 (Kaminogo, M., et al.,Neurol. Med. Chir. (Tokyo), 38(11):704-8; discussion 708-9 (1998)); Therat serine protease inhibitor 2.1 gene (LeCam, A., et al., Biochem.Biophys. Res. Commun., 253(2):311-4 (1998)); A new intracellular serineprotease inhibitor expressed in the rat pituitary gland complexes withgranzyme B (Hill; R. M., et al., FEBS Lett., 440(3):361-4 (1998));3,4-Dichloroisocoumarin (Hammed, A., et al., Proc. Soc. Exp. Biol. Med.,219(2):132-7 (1998)); LEX032 (Bains, A. S., et al., Eur. J. Pharmacol.,356(1):67-72 (1998)); N-tosyl-L-phenylalanine chloromethyl ketone(Dryjanski, M., et al., Biochemistry, 37(40):14151-6 (1998)); Mouse genefor the serine protease inhibitor neuroserpin (P112) (Berger, P., etal., Gene, 214(1-2):25-33 (1998)); Rat serine protease inhibitor 2.3gene (Paul, C., et al., Eur. J. Biochem., 254(3):538-46 (1998)); Ecotin(Yang, S. Q., et al., J. Mol. Biol., 279(4):945-57 (1998)); A 14 kDaplant-related serine protease inhibitor (Roch, P., et al., Dev. Comp.Immunol., 22(1):1-12 (1998)); Matrix-associated serine proteaseinhibitor TFPI-2/33 kDa MSPI (Rao, C. N., et al., Int. J. Cancer,76(5):749-56 (1998)); ONO-3403 (Hiwasa, T., et al., Cancer Lett.,126(2):221-5 (1998)); Bdellastasin (Moser, M., et al., Eur. J. Biochem.,253(1):212-20 (1998)); Bikunin (Xu, Y., et al., J. Mol. Biol.,276(5):955-66 (1998)); Nafamostat mesilate (Mellgren, K., et al.,Thromb. Haemost., 79(2):342-7 (1998)); The growth hormone dependentserine protease inhibitor, Spi 2.1 (Maake, C., et al., Endocrinology,138(12):5630-6 (1997)); Growth factor activator inhibitor type 2, aKunitz-type serine protease inhibitor (Kawaguchi, T., et al., J. Biol.Chem., 272(44):27558-64 (1997)); Heat-stable serine protease inhibitorprotein from ovaries of the desert locust, Schistocerga gregaria(Hamdaoui, A., et al., Biochem. Biophys. Res. Commun., 238(2):357-60(1997)); Bikunin, (Delaria, K. A., et al., J. Biol. Chem.,272(18):12209-14 (1997)); Human placental bikunin (Marlor, C. W., etal., J. Biol. Chem., 272(10):12202-8 (1997)); Hepatocyte growth factoractivator inhibitor, a novel Kunitz-type serine protease inhibitor(Shimomura, T., et al., J. Biol. Chem., 272(10):6370-6 (1997)); FUT-187,oral serine protease inhibitor, (Shiozaki, H., et al., Gan To KagukuRyoho, 23(14): 197 1-9 (1996)); Extracellular matrix-associated serineprotease inhibitors (Mr 33,000, 31,000, and 27,000 (Rao, C. N., et al.,Arch. Biochem. Biophys., 335(1):82-92 (1996)); An irreversibleisocoumarin serine protease inhibitor (Palencia, D. D., et al., Biol.Reprod., 55(3):536-42 (1996)); 4-(2-aminoethyl)-benzenesulfonyl fluoride(AEBSF) (Nakabo, Y., et al., J. Leukoc. Biol., 60(3):328-36 (1996));Neuroserpin (Osterwalder, T., et al., EMBO J., 15(12):2944-53 (1996));Human serine protease inhibitor alpha-1-antitrypsin (Forney, J. R., etal., J. Parasitol., 82(3):496-502 (1996)); Rat serine protease inhibitor2.3 (Simar-Blanchet, A. E., et al., Eur. J. Biochem., 236(2):638-48(1996)); Gebaxate mesilate (parodi, F., et al., J. Cardiothorac. Vasc.Anesth., 10(2):235-7 (1996)); Recombinant serine protease inhibitor,CPTI II (Stankiewicz, M., et al., (Acta Biochim. Pol., 43(3):525-9(1996)); A cysteine-rich serine protease inhibitor (Guamerin II) (Kim,D. R., et al., J. Enzym. Inhib., 10(2):81-91 (1996));Diisopropylfluorophosphate (Lundqvist, H., et al., Inflamm. Res.,44(12):510-7 (1995)); Nexin 1 (Yu, D. W., et al., J. Cell Sci., 108(Pt12):3867-74 (1995)); LEX032 (Scalia, R., et al., Shock, 4(4):251-6(1995)); Protease nexin I (Houenou, L. J., et al., Proc. Natl. Acad.Sci. U.S.A., 92(3):895-9 (1995)); Chymase-directed serine proteaseinhibitor (Woodard S. L., et al., J. Immunol., 153(11):5016-25 (1994));N-alpha-tosyl-L-lysyl-chloromethyl ketone (TLCK) (Bourinbaiar, A. S., etal., Cell Immunol., 155(1):230-6 (1994)); Smpi56 (Ghendler, Y., et al.,Exp. Parasitol., 78(2):121-31 (1994)); Schistosoma haematobium serineprotease (Blanton, R. E., et al., Mol. Biochem. Parasitol., 63(1):1-11(1994)); Spi-1 (Warren, W. C., et al., Mol. Cell Endocrinol.,98(1):27-32 (1993)); TAME (Jessop, J. J., et al., Inflammation,17(5):613-31 (1993)); Antithrombin III (Kalaria, R. N., et al., Am. J.Pathol., 143(3):886-93 (1993)); FOY-305 (Ohkoshi, M., et al., AnticancerRes., 13(4):963-6 (1993)); Camostat mesilate (Senda, S., et al., Intern.Med., 32(4):350-4 (1993)); Pigment epithelium-derived factor (Steele, F.R., et al., Proc. Natl. Acad. Sci. U.S.A., 90(4):1526-30 (1993));Antistasin (Holstein, T. W., et al., FEBS Lett., 309(3):288-92 (1 92));The vaccinia virus K2L gene encodes a serine protease inhibitor (Zhou,J., et al., Virology, 189(2):678-86 (1992)); Bowman-Birk serine-proteaseinhibitor (Werner, M. H., et al., J. Mol. Biol., 225(3):873-89 (1992);FUT-175 (Yanamoto, H., et al., Neurosurgery, 30(3):358-63 (1992));FUT-175; (Yanamoto, H., et al., Neurosurgery, 30(3):351-6, discussion356-7 (1992)); PAI-I (Yreadwell, B. V., et al., J. Orthop. Res.,9(3):309-16 (1991)); 3,4-Dichloroisocoumarin (Rusbridge, N. M., et al.,FEBS Lett., 268(1):133-6 (1990)); Alpha 1-antichymotrypsin (Lindmark, B.E., et al., Am. Rev. Respir. Des., 141(4 Pt 1):884-8 (1990));P-toluenesulfonyl-L-arginine methyl ester (TAME) (Scuderi, P., J.Immunol., 143(1):168-73 (1989)); Aprotinin (Seto, S., et al., Adv. Exp.Med. Biol., 247B:49-54 (1989)); Alpha 1-antichymotrypsin (Abraham, C.R., et al., Cell, 52(4):487-501 (1988)); Contrapsin (Modha, J., et al.,Parasitology, 96(Pt 1):99-109 (1988)); (FOY-305) (Yamauchi, Y., et al.,Hiroshima J. Med. Sci., 36(1):81-7 No abstract available (1987)); Alpha2-antiplasmin (Holmes, W. E., et al., J. Biol. Chem., 262(4):1659-64(1987)); 3,4-dichloroisocoumarin (Harper, J. W., et al., Biochemistry,24(8):1831-41 (1985)); Diisoprophylfluorophosphate (Tsutsui, K., et al.,Biochem. Biophys. Res. Commun., 123(1):271-7 (1984)); Gabexate mesilate(Hesse, B., et al., Pharmacol. Res. Commun., 16(7):637-45 (1984));Phenyl methyl sulfonyl fluoride (Dufer, J., et al., Scand. J. Haematol.,32(1):25-32 (1984)); Aprotinin (Seto, S., et al., Hypertension,5(6):893-9 (1983)); Protease inhibitor CI-2 (McPhalen, C. A., et al., J.Mol. Biol., 168(2):445-7 (1983)); Phenylmethylsulfonyl fluoride (SekarV., et al., Biochem. Biophys. Res. Commun., 89(2):474-8 (1979)); PGE1(Feinstein, M. D., et al., Prostaglandine, 14(6):1075-93 (1977).

c. Combinatorial Libraries and Other Libraries

The source of compounds for the screening assays, can be libraries,including, but are not limited to, combinatorial libraries. Methods forsynthesizing combinatorial libraries and characteristics of suchcombinatorial libraries are known in the art (See generally,Combinatorial Libraries: Synthesis, Screening and Application Potential(Cortese Ed.) Walter de Gruyter, Inc., 1995; Tietze and Lieb, Curr.Opin. Chem. Biol., 2(3):363-71 (1998); Lam, Anticancer Drug Des.,12(3):145-67 (1997); Blaney and Martin, Curr. Opin. Chem. Biol.,1(1):54-9 (1997); and Schultz and Schultz, Biotechnol. Prog.,12(6):729-43 (1996)).

Methods and strategies for generating diverse libraries, primarilypeptide- and nucleotide-based oligomer libraries, have been developedusing molecular biology methods and/or simultaneous chemical synthesismethodologies (see, e.g., Dower et al., Annu. Rep. Med. Chem.,26:271-280 (1991); Fodor et al., Science, 251:767-773 (1991); Jung etal., Angew. Chem. Ind. Ed. Engl., 31:367-383 (1992); Zuckerman et al.,Proc. Natl. Acad. Sci. USA, 89:4505-4509 (1992); Scott et al., Science,249:386-390 (1990); Devlin et al., Science, 249:404-406 (1990); Cwirlaet al., Proc. Natl. Acad. Sci. USA, 87:6378-6382 (1990); and Gallop etal., J. Medicinal Chemistry, 37:1233-1251 (1994)). The resultingcombinatorial libraries potentially contain millions of compounds andthat can be screened to identify compounds that exhibit a selectedactivity.

The libraries fall into roughly three categories:fusion-protein-displayed peptide libraries in which random peptides orproteins are presented on the surface of phage particles or proteinsexpressed from plasmids; support-bound synthetic chemical libraries inwhich individual compounds or mixtures of compounds are presented oninsoluble matrices, such as resin beads (see, e.g., Lam et al., Nature,354:82-84 (1991)) and cotton supports (see, e.g., Eichler et al.,Biochemistry 32:11035-11041 (1993)); and methods in which the compoundsare used in solution (see, e.g., Houghten et al., Nature, 354:84-86(1991); Houghten et al., BioTechniques, 313:412-421 (1992); and Scott etal., Curr. Opin. Biotechnol., 5:40-48 (1994)). There are numerousexamples of synthetic peptide and oligonucleotide combinatoriallibraries and there are many methods for producing libraries thatcontain non-peptidic small organic molecules. Such libraries can bebased on basis set of monomers that are combined to form mixtures ofdiverse organic molecules or that can be combined to form a librarybased upon a selected pharmacophore monomer.

Either a random or a deterministic combinatorial library can be screenedby the presently disclosed and/or claimed screening methods. In eitherof these two libraries, each unit of the library is isolated and/orimmobilized on a solid support. In the deterministic library, one knowsa priori a particular unit's location on each solid support. In a randomlibrary, the location of a particular unit is not known a priorialthough each site still contains a single unique unit. Many methods forpreparing libraries are known to those of skill in this art (see, e.g.,Geysen et al., Proc. Natl. Acad. Sci. USA, 81:3998-4002 (1984), Houghtenet al., Proc. Natl. Acad. Sci. USA, 81:5131-5135 (1985)). Combinatoriallibrary generated by the any techniques known to those of skill in theart are contemplated (see, e.g., Table 1 of Schultz and Schultz,Biotechnol. Prog., 12(6):729-43 (1996)) for screening; Bartel et al.,Science, 261:1411-1418 (1993); Baumbach et al. BioPharm, (May): 24-35(1992); Bock et al. Nature, 355:564-566 (1992); Borman, S.,Combinatorial chemists focus on samll molecules molecular recognition,and automation, Chem. Eng. News, 2(12):29 (1996); Boublik, et al.,Eukaryotic Virus Display: Engineering the Major Surface Glycoproteins ofthe Autographa California Nuclear Polyhedrosis Virus (ACNPV) for thePresentation of Foreign Proteins on the Virus Surface, Bio/Technology,13:1079-1084 (1995); Brenner, et al., Encoded Combinatorial Chemistry,Proc. Natl. Acad Sci. U.S.A., 89:5381-5383 (1992); Caflisch, et al.,Computational Combinatorial Chemistry for De Novo Ligand Design: Reviewand Assessment, Perspect. Drug Discovery Des., 3:51-84 (1995); Cheng, etal., Sequence-Selective Peptide Binding with aPeptido-A,B-trans-steroidal Receptor Selected from an EncodedCombinatorial Library, J. Am. Chem. Soc., 118:1813-1814 (1996); Chu, etal., Affinity Capillary Electrophoresis to Identify the Peptide in APeptide Library that Binds Most Tightly to Vancomycin, J. Org. Chem.,58:648-652 (1993); Clackson, et al., Making Antibody Fragments UsingPhage Display Libraries, Nature, 352:624-628 (1991); Combs, et al.,Protein Structure-Based Combinatorial Chemistry: Discovery ofNon-Peptide Binding Elements to Src SH3 Domain, J. Am. Chem. Soc.,118:287-288 (1996); Cwirla, et al., Peptides On Phage: A Vast Library ofPeptides for Identifying Ligands, Proc. Natl. Acad. Sci. U.S.A.,87:6378-6382 (1990); Ecker, et al., Combinatorial Drug Discovery: WhichMethod will Produce the Greatest Value, Bio/Technology, 13:351-360(1995); Ellington, et al., In vitro Selection of RNA Molecules That BindSpecific Ligands, Nature, 346:818-822 (1990); Ellman, J. A., Variants ofBenzodiazephines, J. Am. Chem. Soc., 114:10997 (1992); Erickson, et al.,The Proteins; Neurath, H., Hill, R. L., Eds.: Academic: New York, 1976;pp. 255-257; Felici, et al., J. Mol. Biol., 222:301-310 (1991); Fodor,et al., Light-Directed, Spatially Addressable Parallel ChemicalSynthesis, Science, 251:767-773 (1991); Francisco, et al., Transport andAnchoring of Beta-Lactamase to the External Surface of E. Coli., Proc.Natl. Acad. Sci. U.S.A., 89:2713-2717 (1992); Georgiou, et al.,Practical Applications of Engineering Gram-Negative Bacterial CellSurfaces, TIBTECH,11:6-10 (1993); Geysen, et al., Use of peptidesynthesis to probe viral antigens for epitopes to a resolution of asingle amino acid, Proc. Natl. Acad. Sci. U.S.A., 81:3998-4002 (1984);Glaser, et al., Antibody Engineering by Condon-Based Mutagenesis in aFilamentous Phage Vector System, J. Immunol., 149:3903-3913 (1992);Gram, et al., In vitro selection and affinity maturation of antibodiesfrom a naive combinatorial immunoglobulin library, Proc. Natl. Acad.Sci., 89:3576-3580 (1992); Han, et al., Liquid-Phase CombinatorialSynthesis, Proc. Natl. Acad. Sci. U.S.A., 92:6419-6423 (1995);Hoogenboom, et al., Multi-Subunit Proteins on the

Surface of Filamentous Phage: Methodologies for Displaying Antibody(Fab) Heavy and Light Chains, Nucleic Acids Res., 19:4133-4137 (1991);Houghten, et al., General Method for the Rapid Solid-Phase Synthesis ofLarge Numbers of Peptides: Specificity of Antigen-Antibody Interactionat the Level of Individual Amino Acids, Proc. Natl. Acad. Sci. U.S.A.,82:5131-5135 (1985); Houghten, et al., The Use of

Synthetic Peptide Combinatorial Libraries for the Determination ofPeptide Ligands in Radio-Receptor Assays-Opiod-Peptides, Bioorg. Med.Chem. Lett., 3:405-412 (1993); Houghten, et al., Generation and Use ofSynthetic Peptide Combinatorial Libraries for Basic Research and DrugDiscovery, Nature, 354:84-86 (1991); Huang, et al., Discovery of NewLigand Binding Pathways in Myoglobin by Random Mutagenesis, NatureStruct. Biol., 1:226-229 (1994); Huse, et al., Generation of a LargeCombinatorial Library of the Immunoglobulin Repertoire In Phage Lambda,Science, 246:1275-1281 (1989); Janda, K. D., New Strategies for theDesign of Catalytic Antibodies, Biotechnol. Prog., 6:178-181 (1990);Jung, et al., Multiple Peptide Synthesis Methods and Their Applications,Angew. Chem. Int. Ed. Engl., 31:367-486 (1992); Kang, et al., Linkage ofRecognition and Replication Functions By Assembling CombinatorialAntibody Fab Libraries Along Phage Surfaces, Proc. Natl. Acad. Sci.U.S.A., 88:4363-4366 (1991a); Kang, et al., Antibody Redesign by ChainShuffling from Random Combinatorial Immunoglobulin Libraries, Proc.Natl. Acad. Sci. U.S.A., 88:11120-11123 (1991b); Kay, et al., An M13Phage Library Displaying Random 38-Amino-Acid-Peptides as a Source ofNovel Sequences with Affinity to Selected Targets Genes, Gene, 128:59-65(1993); Lam, et al., A new type of synthetic peptide library foridentifying ligand-binding activity, Nature, 354:82-84 (1991) (publishederrata apear in Nature, 358:434 (1992) and Nature, 360:768 (1992); Lebl,et al., One Bead One Structure Combinatorial Libraries, Biopolymers(Pept. Sci.), 37:177-198 (1995); Lerner, et al., Antibodies withoutImmunization, Science, 258:1313-1314 (1992); Li, et al., Minimization ofa Polypeptide Hormone, Science, 270:1657-1660 (1995); Light, et al.,Display of Dimeric Bacterial Alkaline Phosphatase on the Major CoatProtein of Filamentous Bacteriophage, Bioorg. Med. Chem. Lett.,3:1073-1079 (1992); Little, et al., Bacterial Surface Presentation ofProteins and Peptides: An Alternative to Phage Technology, TrendsBiotechnol., 11:3-5 (1993); Marks, et al., By-Passing Immunization.Human Antibodies from V-Gene Libraries Displayed on Phage, J. Mol.Biol., 222:581-597 (1991); Matthews, et al., Substrate Phage: Selectionof Protease Substrates by Monovalent Phage Display, Science,260:1113-1117 (1993); McCafferty, et al., Phage Enzymes: Expression andAffinity Chromatography of Functional Alkaline Phosphatase on theSurface of Bacteriophage, Protein Eng., 4:955-961 (1991); Menger, etal., Phosphatase Catalysis Developed Via Combinatorial OrganicChemistry, J. Org. Chem., 60:6666-6667 (1995); Nicolaou, et al., Angew.Chem. Int. Ed. Engl., 34:2289-2291 (1995); Oldenburg, et al., PeptideLigands for A Sugar-Binding Protein Isolated from a Random PeptideLibrary, Proc. Natl. Acad. Sci. U.S.A., 89:5393-5397 (1992); Parmley, etal., Antibody-Selectable Filamentous fd Phage Vectors: AffinityPurification of Target Genes, Genes, 73:305-318 (1988); Pinilla, et al.,Synthetic Peptide Combinatorial Libraries (SPCLS)—Identification of theAntigenic Determinant of Beta-Endorphin Recognized by MonoclonalAntibody-3E7, Gene, 128:71-76 (1993); Pinilla, et al., Review of theUtility of Soluble Combinatorial Libraries, Biopolymers, 37:221-240(1995); Pistor, et al., Expression of Viral Hemegglutinan On the Surfaceof E. Coli., Klin. Wochenschr., 66:110-116 (1989); Pollack, et al.,Selective Chemical Catalysis by an Antibody, Science, 234:1570-1572(1986); Rigler, et al., Fluorescence Correlations, Single MoleculeDetection and Large Number Screening: Applications in Biotechnology, J.Biotechnol., 41:177-186 (1995); Sarvetnick, et al., Increasing theChemical Potential of the Germ-Line Antibody Repertoire, Proc. Natl.Acad. Sci. U.S.A., 90:4008-4011 (1993); Sastry, et al., Cloning of theImmunological Repertiore in Escherichia Coli for Generation ofMonoclonal Catalytic Antibodies: Construction of a Heavy Chain VariableRegion-Specific cDNA Library, Proc. Natl. Acad. Sci. U.S.A.,86:5728-5732 (1989); Scott, et al., Searching for Peptide Ligands withan Epitope Library, Science, 249:386-390 (1990); Sears, et al.,Engineering Enzymes for Bioorganic Synthesis: Peptide Bond Formation,Biotechnol. Prog., 12:423-433 (1996); Simon, et. al., Peptides: AModular Approach to Drug Discovery, Proc. Natl. Acad. Sci. U.S.A.,89:9367-9371 (1992); Still, et al., Discovery of Sequence-SelectivePeptide Binding by Synthetic Receptors Using Encoded CombinatorialLibraries, Acc. Chem. Res., 29:155-163 (1996); Thompson, et al.,Synthesis and Applications of Small Molecule Libraries, Chem. Rev.,96:555-600 (1996); Tramontano, et al., Catalytic Antibodies, Science,234:1566-1570 (1986); Wrighton, et al., Small Peptides as PotentMimetics of the Protein Hormone Erythropoietin, Science, 273:458-464(1996); York, et al., Combinatorial mutagenesis of the reactive siteregion in plasminogen activator inhibitor I, J. Biol. Chem.,266:8595-8600 (1991); Zebedee, et al., Human Combinatorial AntibodyLibraries to Hepatitis B Surface Antigen, Proc. Natl. Acad. Sci. U.S.A.,89:3175-3179 (1992); Zuckerman, et al., Identification ofHighest-Affinity Ligands by Affinity Selection from Equimolar PeptideMixtures Generated by Robotic Synthesis, Proc. Natl. Acad. Sci. U.S.A.,89:4505-4509 (1992).

For example, peptides that bind to an MTSP protein or a protease domainof an MTSP protein can be identified using phage display libraries. Inan exemplary embodiment, this method can include a) contacting phagefrom a phage library with the MTSP protein or a protease domain thereof;(b) isolating phage that bind to the protein; and (c) determining theidentity of at least one peptide coded by the isolated phage to identifya peptide that binds to an MTSP protein.

H. MODULATORS OF THE ACTIVITY OF MTSP PROTEINS

Provided herein are compounds, identified by screening or produced usingthe MTSP proteins or protease domain in other screening methods, thatmodulate the activity of an MTSP. These compounds act by directlyinteracting with the MTSP protein or by altering transcription ortranslation thereof. Such molecules include, but are not limited to,antibodies that specifically react with an MTSP protein, particularlywith the protease domain thereof, antisense nucleic acids that alterexpression of the MTSP protein, antibodies, peptide mimetics and othersuch compounds.

1. Antibodies

Antibodies, including polyclonal and monoclonal antibodies, thatspecifically bind to the MTSP proteins provided herein, particularly tothe single chain protease domains thereof are provided. Preferably, theantibody is a monoclonal antibody, and preferably, the antibodyspecifically binds to the protease domain of the MTSP protein. Inparticular embodiments, antibodies to each of the single chain ofprotease domain of MTSP1, MTSP3, MTSP4 and MTSP6. Also provided areantibodies that specifically bind to any domain of MTSP3 or MTSP4, andto double chain forms thereof.

The MTSP protein and domains, fragments, homologs and derivativesthereof may be used as immunogens to generate antibodies thatspecifically bind such immunogens. Such antibodies include but are notlimited to polyclonal, monoclonal, chimeric, single chain, Fabfragments, and an Fab expression library. In a specific embodiment,antibodies to human MTSP protein are produced. In another embodiment,complexes formed from fragments of MTSP protein, which fragments containthe serine protease domain, are used as immunogens for antibodyproduction.

Various procedures known in the art may be used for the production ofpolyclonal antibodies to MTSP protein, its domains, derivatives,fragments or analogs. For production of the antibody, various hostanimals can be immunized by injection with the native MTSP protein or asynthetic version, or a derivative of the foregoing, such as across-linked MTSP protein. Such host animals include but are not limitedto rabbits, mice, rats, etc. Various adjuvants can be used to increasethe immunological response, depending on the host species, and includebut are not limited to Freund's (complete and incomplete), mineral gelssuch as aluminum hydroxide, surface active substances such aslysolecithin, pluronic polyols, polyanions, peptides, oil emulsions,dinitrophenol, and potentially useful human adjuvants such as bacilleCalmette-Guerin (BCG) and corynebacterium parvum.

For preparation of monoclonal antibodies directed towards an MTSPprotein or domains, derivatives, fragments or analogs thereof, anytechnique that provides for the production of antibody molecules bycontinuous cell lines in culture may be used. Such techniques includebut are not restricted to the hybridoma technique originally developedby Kohler and Milstein (Nature 256:495-497 (1975)), the triomatechnique, the human B-cell hybridoma technique (Kozbor et al.,Immunology Today 4:72 (1983)), and the EBV hybridoma technique toproduce human monoclonal antibodies (Cole et al., in MonoclonalAntibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96 (1985)). Inan additional embodiment, monoclonal antibodies can be produced ingerm-free animals utilizing recent technology (PCT/US90/02545). Humanantibodies may be used and can be obtained by using human hybridomas(Cote et al., Proc. Natl. Acad. Sci. USA 80:2026-2030 (1983)). Or bytransforming human B cells with EBV virus in vitro (Cole et al., inMonoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96(1985)). Techniques developed for the production of “chimericantibodies” (Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855(1984); Neuberger et al., Nature 312:604-608 (1984); Takeda et al.,Nature 314:452-454 (1985)) by splicing the genes from a mouse antibodymolecule specific for the MTSP protein together with genes from a humanantibody molecule of appropriate biological activity can be used.

Techniques described for the production of single chain antibodies (U.S.Pat. No. 4,946,778) can be adapted to produce MTSP protein-specificsingle chain antibodies. An additional embodiment uses the techniquesdescribed for the construction of Fab expression libraries (Huse et al.,Science 246:1275-1281 (1989)) to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity for MTSP proteinor MTSP protein, or domains, derivatives, or analogs thereof Non-humanantibodies can be “humanized” by known methods (see, e.g., U.S. Pat. No.5,225,539).

Antibody fragments that contain the idiotypes of MTSP protein can begenerated by techniques known in the art. For example, such fragmentsinclude but are not limited to: the F(ab′)2 fragment which can beproduced by pepsin digestion of the antibody molecule; the Fab′fragments that can be generated by reducing the disulfide bridges of theF(ab′)2 fragment, the Fab fragments that can be generated by treatingthe antibody molecular with papain and a reducing agent, and Fvfragments.

In the production of antibodies, screening for the desired antibody canbe accomplished by techniques known in the art, e.g., ELISA(enzyme-linked immunosorbent assay). To select antibodies specific to aparticular domain of the MTSP protein one may assay generated hybridomasfor a product that binds to the fragment of the MTSP protein thatcontains such a domain.

The foregoing antibodies can be used in methods known in the artrelating to the localization and/or quantitation of MTSP proteins, e.g.,for imaging these proteins, measuring levels thereof in appropriatephysiological samples, in diagnostic methods, etc.

In another embodiment, (see infra), anti-MTSP protein antibodies, orfragments thereof, containing the binding domain are used as therapeuticagents.

2. Peptides and Peptide Mimetics

Provided herein are methods for identifying molecules that bind to andmodulate the activity of MTSP proteins. Included among molecules thatbind to MTSPs, particularly the single chain protease domain orcatalytically active fragments thereof, are peptides and peptidemimetics. Peptide mimetics are molecules or compounds that mimic thenecessary molecular conformation of a ligand or polypeptide for specificbinding to a target molecule such as, e.g., an MTSP protein. In anexemplary embodiment, the peptides or peptide mimetics bind to theprotease domain of the MTSP protein. Such peptides and peptide mimeticsinclude those of antibodies that specifically bind an MTSP protein and,preferably, bind to the protease domain of an MTSP protein. The peptidesand peptide mimetics identified by methods provided herein can beagonists or antagonists of MTSP proteins.

Such peptides and peptide mimetics are useful for diagnosing, treating,preventing, and screening for a disease or disorder associated with MTSPprotein activity in a mammal. In addition, the peptides and peptidemimetics are useful for identifying, isolating, and purifying moleculesor compounds that modulate the activity of an MTSP protein, orspecifically bind to an MTSP protein, preferably, the protease domain ofan MTSP protein. Low molecular weight peptides and peptide mimetics canhave strong binding properties to a target molecule, e.g., an MTSPprotein or, preferably, to the protease domain of an MTSP protein.

Peptides and peptide mimetics that bind to MTSP proteins as describedherein can be administered to mammals, including humans, to modulateMTSP protein activity. Thus, methods for therapeutic treatment andprevention of neoplastic diseases comprise administering a peptide orpeptide mimetic compound in an amount sufficient to modulate suchactivity are provided. Thus, also provided herein are methods fortreating a subject having such a disease or disorder in which a peptideor peptide mimetic compound is administered to the subject in atherapeutically effective dose or amount.

Compositions containing the peptides or peptide mimetics can beadministered for prophylactic and/or therapeutic treatments. Intherapeutic applications, compositions can be administered to a patientalready suffering from a disease, as described above, in an amountsufficient to cure or at least partially arrest the symptoms of thedisease and its complications. Amounts effective for this use willdepend on the severity of the disease and the weight and general stateof the patient.

In prophylactic applications, compositions containing the peptides andpeptide mimetics are administered to a patient susceptible to orotherwise at risk of a particular disease. Such an amount is defined tobe a “prophylactically effective dose”. In this use, the precise amountsagain depend on the patient's state of health and weight.

Accordingly, the peptides and peptide mimetics that bind to an MTSPprotein can be used generating pharmaceutical compositions containing,as an active ingredient, at least one of the peptides or peptidemimetics in association with a pharmaceutical carrier or diluent. Thecompounds can be administered, for example, by oral, pulmonary, parental(intramuscular, intraperitoneal, intravenous (IV) or subcutaneousinjection), inhalation (via a fine powder formulation), transdermal,nasal, vaginal, rectal, or sublingual routes of administration and canbe formulated in dosage forms appropriate for each route ofadministration (see, e.g., International PCT application Nos. WO93/25221 and WO 94/17784; and European Patent Application 613,683).

Peptides and peptide mimetics that bind to MTSP proteins are useful invitro as unique tools for understanding the biological role of MTSPproteins, including the evaluation of the many factors thought toinfluence, and be influenced by, the production of MTSP protein. Suchpeptides and peptide mimetics are also useful in the development ofother compounds that bind to and modulate the activity of an MTSPprotein, because such compounds provide important information on therelationship between structure and activity that should facilitate suchdevelopment.

The peptides and peptide mimetics are also useful as competitive bindersin assays to screen for new MTSP proteins or MTSP protein agonists. Insuch assay embodiments, the compounds can be used without modificationor can be modified in a variety of ways; for example, by labeling, suchas covalently or non-covalently joining a moiety which directly orindirectly provides a detectable signal. In any of these assays, thematerials thereto can be labeled either directly or indirectly.Possibilities for direct labeling include label groups such as:radiolabels such as ¹²⁵I enzymes (U.S. Pat. No. 3,645,090) such asperoxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat.No. 3,940,475) capable of monitoring the change in fluorescenceintensity, wavelength shift, or fluorescence polarization. Possibilitiesfor indirect labeling include biotinylation of one constituent followedby binding to avidin coupled to one of the above label groups. Thecompounds may also include spacers or linkers in cases where thecompounds are to be attached to a solid support.

Moreover, based on their ability to bind to an MTSP protein, thepeptides and peptide mimetics can be used as reagents for detecting MTSPproteins in living cells, fixed cells, in biological fluids, in tissuehomogenates, in purified, natural biological materials, etc. Forexample, by labelling such peptides and peptide mimetics, one canidentify cells having MTSP proteins. In addition, based on their abilityto bind an MTSP protein, the peptides and peptide mimetics can be usedin situ staining, FACS (fluorescence-activated cell sorting), Westernblotting, ELISA, etc. In addition, based on their ability to bind to anMTSP protein, the peptides and peptide mimetics can be used inpurification of MTSP protein polypeptides or in purifying cellsexpressing the MTSP protein polypeptides, e.g., a polypeptide encodingthe protease domain of an MTSP protein.

The peptides and peptide mimetics can also be used as commercialreagents for various medical research and diagnostic uses.

The activity of the peptides and peptide mimetics can be evaluatedeither in vitro or in vivo in one of the numerous models described inMcDonald (1992) Am. J. of Pediatric Hematology/Oncology, 14:8-21, whichis incorporated herein by reference.

3. Peptide and Peptide Mimetic Therapy

Peptides and peptide mimetics that can bind to MTSP proteins or theprotease domain of MTSP proteins and modulate the activity thereof, orhave MTSP protein activity, can be used for treatment of neoplasticdiseases. The peptides and peptide mimetics may be delivered, in vivo orex vivo, to the cells of a subject in need of treatment. Further,peptides which have MTSP protein activity can be delivered, in vivo orex vivo, to cells which carry mutant or missing alleles encoding theMTSP protein gene. Any of the techniques described herein or known tothe skilled artisan can be used for preparation and in vivo or ex vivodelivery of such peptides and peptide mimetics that are substantiallyfree of other human proteins. For example, the peptides can be readilyprepared by expression in a microorganism or synthesis in vitro.

The peptides or peptide mimetics can be introduced into cells, in vivoor ex vivo, by microinjection or by use of liposomes, for example.Alternatively, the peptides or peptide mimetics may be taken up bycells, in vivo or ex vivo, actively or by diffusion. In addition,extracellular application of the peptide or peptide mimetic may besufficient to effect treatment of a neoplastic disease. Other molecules,such as drugs or organic compounds, that: 1) bind to an MTSP protein orprotease domain thereof; or 2) have a similar function or activity to anMTSP protein or protease domain thereof, may be used in methods fortreatment.

4. Rational Drug Design

The goal of rational drug design is to produce structural analogs ofbiologically active polypeptides or peptides of interest or of smallmolecules or peptide mimetics with which they interact (e.g., agonists,antagonists, inhibitors) in order to fashion drugs which are, e.g., moreactive or stable forms thereof; or which, e.g., enhance or interferewith the function of a polypeptide in vivo (e.g., an MTSP protein). Inone approach, one first determines the three-dimensional structure of aprotein of interest (e.g., an MTSP protein or polypeptide having aprotease domain) or, for example, of a MTSP protein-ligand complex, byX-ray crystallography, by computer modeling or most typically, by acombination of approaches (see, e.g., Erickson et al. 1990). Also,useful information regarding the structure of a polypeptide may begained by modeling based on the structure of homologous proteins. Inaddition, peptides can be analyzed by an alanine scan. In thistechnique, an amino acid residue is replaced by Ala, and its effect onthe peptide's activity is determined. Each of the amino acid residues ofthe peptide is analyzed in this manner to determine the importantregions of the peptide.

Also, a polypeptide or peptide that binds to an MTSP protein or,preferably, the protease domain of an MTSP protein, can be selected by afunctional assay, and then the crystal structure of this polypeptide orpeptide can be determined. The polypeptide can be, for example, anantibody specific for an MTSP protein or the protein domain of an MTSPprotein. This approach can yield a pharmacore upon which subsequent drugdesign can be based. Further, it is possible to bypass thecrystallography altogether by generating anti-idiotypic polypeptides orpeptides, (anti-ids) to a functional, pharmacologically activepolypeptide or peptide that binds to an MTSP protein or protease domainof an MTSP protein. As a mirror image of a mirror image, the bindingsite of the anti-ids is expected to be an analog of the original targetmolecule, e.g., an MTSP protein or polypeptide having an MTSP protein.The anti-id could then be used to identify and isolate peptides frombanks of chemically or biologically produced banks of peptides. Selectedpeptides would then act as the pharmacore.

Thus, one may design drugs which have, e.g., improved activity orstability or which act as modulators (e.g., inhibitors, agonists,antagonists, etc.) of MTSP protein activity, and are useful in themethods, particularly the methods for diagnosis, treatment, prevention,and screening of a neoplastic disease. By virtue of the availability ofcloned MTSP protein sequences, sufficient amounts of the MTSP proteinpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, the knowledge of the amino acidsequence of an MTSP protein or the protease domain thereof, e.g., theprotease domain encoded by the amino acid sequence of SEQ ID NO: 2, canprovide guidance on computer modeling techniques in place of, or inaddition to, X-ray crystallography.

Methods of Identifying Peptides and Peptide Mimetics that Bind to MTSPProteins

Peptides having a binding affinity to the MTSP protein polypeptidesprovided herein (e.g., an MTSP protein or a polypeptide having aprotease domain of an MTSP protein) can be readily identified, forexample, by random peptide diversity generating systems coupled with anaffinity enrichment process. Specifically, random peptide diversitygenerating systems include the “peptides on plasmids” system (see, e.g.,U.S. Pat. Nos. 5,270,170 and 5,338,665); the “peptides on phage” system(see, e.g., U.S. Pat. No. 6,121,238 and Cwirla, et al. (1990) Proc.Natl. Acad. Sci. U.S.A. 87:6378-6382); the “polysome system;” the“encoded synthetic library (ESL)” system; and the “very large scaleimmobilized polymer synthesis” system (see, e.g., U.S. Pat. No.6,121,238; and Dower et al. (1991) An. Rep. Med. Chem. 26:271-280

For example, using the procedures described above, random peptides cangenerally be designed to have a defined number of amino acid residues inlength (e.g., 12). To generate the collection of oligonucleotidesencoding the random peptides, the codon motif (NNK)x, where N isnucleotide A, C, G, or T (equimolar; depending on the methodologyemployed, other nucleotides can be employed), K is G or T (equimolar),and x is an integer corresponding to the number of amino acids in thepeptide (e.g., 12) can be used to specify any one of the 32 possiblecodons resulting from the NNK motif: 1 for each of 12 amino acids, 2 foreach of 5 amino acids, 3 for each of 3 amino acids, and only one of thethree stop codons. Thus, the NNK motif encodes all of the amino acids,encodes only one stop codon, and reduces codon bias.

The random peptides can be presented, for example, either on the surfaceof a phage particle, as part of a fusion protein containing either thepIII or the pVIII coat protein of a phage fd derivative (peptides onphage) or as a fusion protein with the LacI peptide fusion protein boundto a plasmid (peptides on plasmids). The phage or plasmids, includingthe DNA encoding the peptides, can be identified and isolated by anaffinity enrichment process using immobilized MTSP protein polypeptidehaving a protease domain. The affinity enrichment process, sometimescalled “panning,” typically involves multiple rounds of incubating thephage, plasmids, or polysomes with the immobilized MTSP proteinpolypeptide, collecting the phage, plasmids, or polysomes that bind tothe MTSP protein polypeptide (along with the accompanying DNA or mRNA),and producing more of the phage or plasmids (along with the accompanyingLacI-peptide fusion protein) collected.

Characteristics of Peptides and Peptide Mimetics

Typically, the molecular weight of preferred peptides or peptidemimetics is from about 250 to about 8,000 daltons. If the peptides areoligomerized, dimerized and/or derivatized with a hydrophilic polymer(e.g., to increase the affinity and/or activity of the compounds), themolecular weights of such peptides can be substantially greater and canrange anywhere from about 500 to about 120,000 daltons, more preferablyfrom about 8,000 to about 80,000 daltons. Such peptides can comprise 9or more amino acids wherein the amino acids are naturally occurring orsynthetic (non-naturally occurring) amino acids. One skilled in the artwould know how to determine the affinity and molecular weight of thepeptides and peptide mimetics suitable for therapeutic and/or diagnosticpurposes (e.g., see Dower et al., U.S. Pat. No. 6,121,238).

The peptides may be covalently attached to one or more of a variety ofhydrophilic polymers. Suitable hydrophilic polymers include, but are notlimited to, polyalkylethers as exemplified by polyethylene glycol andpolypropylene glycol, polylactic acid, polyglycolic acid,polyoxyalkenes, polyvinylalcohol, polyvinylpyrrolidone, cellulose andcellulose derivatives, dextran and dextran derivatives, etc. When thepeptide compounds are derivatized with such polymers, their solubilityand circulation half-lives can be increased with little, if any,diminishment in their binding activity. The peptide compounds may bedimerized and each of the dimeric subunits can be covalently attached toa hydrophilic polymer. The peptide compounds can be PEGylated, i.e.,covalently attached to polyethylene glycol (PEG).

Peptide analogs are commonly used in the pharmaceutical industry asnon-peptide drugs with properties analogous to those of the templatepeptide. These types of non-peptide compounds are termed “peptidemimetics” or “peptidomimetics” (Luthman et al., A Textbook of DrugDesign and Development, 14:386-406, 2nd Ed., Harwood Academic Publishers(1996); Joachim Grante (1994) Angew. Chem. Int. Ed. Engl., 33:1699-1720;Fauchere (1986) J. Adv. Drug Res., 15:29; Veber and Freidinger (1985)TINS, p. 392; and Evans et al. (1987) J. Med. Chem. 30:1229). Peptidemimetics that are structurally similar to therapeutically usefulpeptides may be used to produce an equivalent or enhanced therapeutic orprophylactic effect. Preparation of peptidomimetics and structuresthereof are known to those of skill in this art.

Systematic substitution of one or more amino acids of a consensussequence with a D-amino acid of the same type (e.g., D-lysine in placeof L-lysine) may be used to generate more stable peptides. In addition,constrained peptides containing a consensus sequence or a substantiallyidentical consensus sequence variation may be generated by methods knownin the art (Rizo et al. (1992) An. Rev. Biochem., 61:387, incorporatedherein by reference); for example, by adding internal cysteine residuescapable of forming intramolecular disulfide bridges which cyclize thepeptide.

Those skilled in the art would appreciate that modifications may be madeto the peptides and mimetics without deleteriously effecting thebiological or functional activity of the peptide. Further, the skilledartisan would know how to design non-peptide structures in threedimensional terms, that mimic the peptides that bind to a targetmolecule, e.g., an MTSP protein or, preferably, the protease domain ofMTSP proteins (see, e.g., Eck and Sprang (1989) J. Biol. Chem., 26:17605-18795).

When used for diagnostic purposes, the peptides and peptide mimetics maybe labeled with a detectable label and, accordingly, the peptides andpeptide mimetics without such a label can serve as intermediates in thepreparation of labeled peptides and peptide mimetics. Detectable labelscan be molecules or compounds, which when covalently attached to thepeptides and peptide mimetics, permit detection of the peptide andpeptide mimetics in vivo, for example, in a patient to whom the peptideor peptide mimetic has been administered, or in vitro, e.g., in a sampleor cells. Suitable detectable labels are well known in the art andinclude, by way of example, radioisotopes, fluorescent labels (e.g.,fluorescein), and the like. The particular detectable label employed isnot critical and is selected relative to the amount of label to beemployed as well as the toxicity of the label at the amount of labelemployed. Selection of the label relative to such factors is well withinthe skill of the art.

Covalent attachment of a detectable label to the peptide or peptidemimetic is accomplished by conventional methods well known in the art.For example, when the ¹²⁵I radioisotope is employed as the detectablelabel, covalent attachment of ¹²⁵I to the peptide or the peptide mimeticcan be achieved by incorporating the amino acid tyrosine into thepeptide or peptide mimetic and then iodinating the peptide (see, e.g.,Weaner et al. (1994) Synthesis and Applications of isotopically LabelledCompounds, pp. 137-140). If tyrosine is not present in the peptide orpeptide mimetic, incorporation of tyrosine to the N or C terminus of thepeptide or peptide mimetic can be achieved by well known chemistry.Likewise, ³²P can be incorporated onto the peptide or peptide mimetic asa phosphate moiety through, for example, a hydroxyl group on the peptideor peptide mimetic using conventional chemistry.

Labeling of peptidomimetics usually involves covalent attachment of oneor more labels, directly or through a spacer (e.g., an amide group), tonon-interfering position(s) on the peptidomimetic that are predicted byquantitative structure-activity data and/or molecular modeling. Suchnon-interfering positions generally are positions that do not formdirect contacts with the macromolecules(s) to which the peptidomimeticbinds to produce the therapeutic effect. Derivatization (e.g., labeling)of peptidomimetics should not substantially interfere with the desiredbiological or pharmacological activity of the peptidomimetic.

6. Methods of Preparing Peptides and Peptide Mimetics

Peptides that bind to MTSP proteins can be prepared by classical methodsknown in the art, for example, by using standard solid phase techniques.The standard methods include exclusive solid phase synthesis, partialsolid phase synthesis methods, fragment condensation, classical solutionsynthesis, and even by recombinant DNA technology (see, e.g., Merrifield(1963) J. Am. Chem. Soc., 85:2149, incorporated herein by reference.)

Using the “encoded synthetic library” or “very large scale immobilizedpolymer synthesis” systems (see, e.g., U.S. Pat. Nos. 5,925,525, and5,902,723); one can not only determine the minimum size of a peptidewith the activity of interest, one can also make all of the peptidesthat form the group of peptides that differ from the preferred motif (orthe minimum size of that motif) in one, two, or more residues. Thiscollection of peptides can then be screened for ability to bind to thetarget molecule, e.g., and MTSP protein or, preferably, the proteasedomain of an MTSP protein. This immobilized polymer synthesis system orother peptide synthesis methods can also be used to synthesizetruncation analogs and deletion analogs and combinations of truncationand deletion analogs of the peptide compounds.

These procedures can also be used to synthesize peptides in which aminoacids other than the 20 naturally occurring, genetically encoded aminoacids are substituted at one, two, or more positions of the peptide. Forinstance, naphthylalanine can be substituted for tryptophan,facilitating synthesis. Other synthetic amino acids that can besubstituted into the peptides include L-hydroxypropyl,L-3,4-dihydroxy-phenylalanyl, d amino acids such as L-d-hydroxylysyl andD-d-methylalanyl, L-α-methylalanyl, β amino acids, and isoquinolyl. Damino acids and non-naturally occurring synthetic amino acids can alsobe incorporated into the peptides (see, e.g., Roberts et al. (1983)Unusual Amino/Acids in Peptide Synthesis, 5(6):341-449).

The peptides may also be modified by phosphorylation (see, e.g., W.Bannwarth et al. (1996) Biorganic and Medicinal Chemistry Letters,6(17):2141-2146), and other methods for making peptide derivatives (see,e.g., Hruby et al. (1990) Biochem. J., 268(2):249-262). Thus, peptidecompounds also serve as a basis to prepare peptide mimetics with similarbiological activity.

Those of skill in the art recognize that a variety of techniques areavailable for constructing peptide mimetics with the same or similardesired biological activity as the corresponding peptide compound butwith more favorable activity than the peptide with respect tosolubility, stability, and susceptibility to hydrolysis and proteolysis(see, e.g., Morgan et al. (1989) An. Rep. Med. Chem., 24:243-252).Methods for preparing peptide mimetics modified at the N-terminal aminogroup, the C-terminal carboxyl group, and/or changing one or more of theamido linkages in the peptide to a non-amido linkage are known to thoseof skill in the art.

Amino terminus modifications include alkylating, acetylating, adding acarbobenzoyl group, forming a succinimide group, etc. (see, e.g., Murrayet al. (1995) Burger's Medicinal Chemistry and Drug Discovery, 5th ed.,Vol. 1, Manfred E. Wolf, ed., John Wiley and Sons, Inc.). C-terminalmodifications include mimetics wherein the C-terminal carboxyl group isreplaced by an ester, an amide or modifications to form a cyclicpeptide.

In addition to N-terminal and C-terminal modifications, the peptidecompounds, including peptide mimetics, can advantageously be modifiedwith or covalently coupled to one or more of a variety of hydrophilicpolymers. It has been found that when peptide compounds are derivatizedwith a hydrophilic polymer, their solubility and circulation half-livesmay be increased and their immunogenicity is masked, with little, ifany, diminishment in their binding activity. Suitable nonproteinaceouspolymers include, but are not limited to, polyalkylethers as exemplifiedby polyethylene glycol and polypropylene glycol, polylactic acid,polyglycolic acid, polyoxyalkenes, polyvinylalcohol,polyvinylpyrrolidone, cellulose and cellulose derivatives, dextran anddextran derivatives, etc. Generally, such hydrophilic polymers have anaverage molecular weight ranging from about 500 to about 100,000daltons, more preferably from about 2,000 to about 40,000 daltons and,even more preferably, from about 5,000 to about 20,000 daltons. Thehydrophilic polymers also can have an average molecular weights of about5,000 daltons, 10,000 daltons and 20,000 daltons.

Methods for derivatizing peptide compounds or for coupling peptides tosuch polymers have been described (see, e.g., Zallipsky (1995)Bioconjugate Chem., 6:150-165; Monfardini et al. (1995) BioconjugateChem., 6:62-69; U.S. Pat. No. 4,640,835; U.S. Pat. No. 4,496,689; U.S.Pat. No. 4,301,144; U.S. Pat. No. 4,670,417; U.S. Pat. No. 4,791,192;U.S. Pat. No. 4,179,337 and WO 95/34326, all of which are incorporatedby reference in their entirety herein).

Other methods for making peptide derivatives are described, for example,in Hruby et al. (1990), Biochem J., 268(2):249-262, which isincorporated herein by reference. Thus, the peptide compounds also serveas structural models for non-peptidic compounds with similar biologicalactivity. Those of skill in the art recognize that a variety oftechniques are available for constructing compounds with the same orsimilar desired biological activity as a particular peptide compound butwith more favorable activity with respect to solubility, stability, andsusceptibility to hydrolysis and proteolysis (see, e.g., Morgan et al.(1989) An. Rep. Med. Chem., 24:243-252, incorporated herein byreference). These techniques include replacing the peptide backbone witha backbone composed of phosphonates, amidates, carbamates, sulfonamides,secondary amines, and N-methylamino acids.

Peptide compounds may exist in a cyclized form with an intramoleculardisulfide bond between the thiol groups of the cysteines. Alternatively,an intermolecular disulfide bond between the thiol groups of thecysteines can be produced to yield a dimeric (or higher oligomeric)compound. One or more of the cysteine residues may also be substitutedwith a homocysteine.

I. CONJUGATES

A conjugate, containing: a) a single chain protease domain (orproteolytically active portion thereof) of an MTSP protein or an MTSP3,MTSP4 or MTSP6 full length zymogen, activated form thereof, or double orsingle chain protease domain thereof; and b) a targeting agent linked tothe MTSP protein directly or via a linker, wherein the agentfacilitates: i) affinity isolation or purification of the conjugate; ii)attachment of the conjugate to a surface; iii) detection of theconjugate; or iv) targeted delivery to a selected tissue or cell, isprovided herein. The conjugate can be a chemical conjugate or a fusionprotein mixture thereof.

The targeting agent is preferably a protein or peptide fragment, such asa tissue specific or tumor specific monoclonal antibody or growth factoror fragment thereof linked either directly or via a linker to an MTSPprotein or a protease domain thereof. The targeting agent may also be aprotein or peptide fragment that contains a protein binding sequence, anucleic acid binding sequence, a lipid binding sequence, apolysaccharide binding sequence, or a metal binding sequence, or alinker for attachment to a solid support. In a particular embodiment,the conjugate contains a) the MTSP or portion thereof, as describedherein; and b) a targeting agent linked to the MTSP protein directly orvia a linker.

Conjugates, such as fusion proteins and chemical conjugates, of the MTSPprotein with a protein or peptide fragment (or plurality thereof) thatfunctions, for example, to facilitate affinity isolation or purificationof the MTSP protein domain, attachment of the MTSP protein domain to asurface, or detection of the MTSP protein domain are provided. Theconjugates can be produced by chemical conjugation, such as via thiollinkages, but are preferably produced by recombinant means as fusionproteins. In the fusion protein, the peptide or fragment thereof islinked to either the N-terminus or C-terminus of the MTSP proteindomain. In chemical conjugates the peptide or fragment thereof may belinked anywhere that conjugation can be effected, and there may be aplurality of such peptides or fragments linked to a single MTSP proteindomain or to a plurality thereof

The targeting agent is preferably for in vitro delivery to a cell ortissue, and includes agents such as cell or tissue-specific antibodies,growth factors and other factors expressed on specific cells; and othercell or tissue specific agents the promote directed delivery of a linkedprotein.

Most preferably the targeting agent specifically delivers the MTSPprotein to selected cells by interaction with a cell surface protein andinternalization of conjugate or MTSP protein portion thereof Theseconjugate are used in a variety of methods and are particularly suitedfor use in methods of activation of prodrugs, such as prodrugs that uponcleavage by the particular MTSP protein are cytotoxic. The prodrugs areadministered prior to simultaneously with or subsequently to theconjugate. Upon delivery to the targeted cells, the protease activatesthe prodrug, which then exhibits is therapeutic effect, such as acytotoxic effect.

1. Conjugation

Conjugates with linked MTSP protein domains can be prepared either bychemical conjugation, recombinant DNA technology, or combinations ofrecombinant expression and chemical conjugation. The MTSP proteindomains and the targeting agent may be linked in any orientation andmore than one targeting agents and/or MTSP protein domains may bepresent in a conjugate.

a. Fusion Proteins

Fusion proteins are proved herein. A fusion protein contains: a) one ora plurality of domains of an MTSP proteins and b) a targeting agent. Thefusion proteins are preferably produced by recombinant expression ofnucleic acids that encode the fusion protein.

b. Chemical Conjugation

To effect chemical conjugation herein, the MTSP protein domain is linkedvia one or more selected linkers or directly to the targeting agent.Chemical conjugation must be used if the targeted agent is other than apeptide or protein, such a nucleic acid or a non-peptide drug. Any meansknown to those of skill in the art for chemically conjugating selectedmoieties may be used.

2. Linkers

Linkers for two purposes are contemplated herein. The conjugates mayinclude one or more linkers between the MTSP protein portion and thetargeting agent. Additionally, linkers are used for facilitating orenhancing immobilization of an MTSP protein or portion thereof on asolid support, such as a microtiter plate, silicon or silicon-coatedchip, glass or plastic support, such as for high throughput solid phasescreening protocols.

Any linker known to those of skill in the art for preparation ofconjugates may be used herein. These linkers are typically used in thepreparation of chemical conjugates; peptide linkers may be incorporatedinto fusion proteins.

Linkers can be any moiety suitable to associate a domain of MTSP proteinand a targeting agent. Such linkers and linkages include, but are notlimited to, peptidic linkages, amino acid and peptide linkages,typically containing between one and about 60 amino acids, moregenerally between about 10 and 30 amino acids, chemical linkers, such asheterobifunctional cleavable cross-linkers, including but are notlimited to, N-succinimidyl (4-iodoacetyl)-aminobenzoate,sulfosuccinimidyl (4-iodoacetyl)-aminobenzoate,4-succinimidyl-oxycarbonyl-a-(2-pyridyldithio)toluene,sulfosuccinimidyl-6-[a-methyl-a-(pyridyldithiol)-toluamido]hexanoate,N-succinimidyl-3-(-2-pyridyldithio)-propionate, succinimidyl6[3(-(-2-pyridyldithio)-proprionamido]hexanoate, sulfosuccinimidyl6[3(-(-2-pyridyldithio)-propionamido]hexanoate,3-(2-pyridyldithio)-propionyl hydrazide, Ellman's reagent,dichlorotriazinic acid, and S-(2-thiopyridyl)-L-cysteine. Other linkersinclude, but are not limited to peptides and other moieties that reducestearic hindrance between the domain of MTSP protein and the targetingagent, intracellular enzyme substrates, linkers that increase theflexibility of the conjugate, linkers that increase the solubility ofthe conjugate, linkers that increase the serum stability of theconjugate, photocleavable linkers and acid cleavable linkers.

Other exemplary linkers and linkages that are suitable for chemicallylinked conjugates include, but are not limited to, disulfide bonds,thioether bonds, hindered disulfide bonds, and covalent bonds betweenfree reactive groups, such as amine and thiol groups. These bonds areproduced using heterobifunctional reagents to produce reactive thiolgroups on one or both of the polypeptides and then reacting the thiolgroups on one polypeptide with reactive thiol groups or amine groups towhich reactive maleimido groups or thiol groups can be attached on theother. Other linkers include, acid cleavable linkers, such asbismaleimideothoxy propane, acid labile-transferrin conjugates andadipic acid dihydrazide, that would be cleaved in more acidicintracellular compartments; cross linkers that are cleaved upon exposureto UV or visible light and linkers, such as the various domains, such asC_(H)1, C_(H)2, and C_(H)3, from the constant region of human IgG₁ (see,Batra et al. Molecular Immunol., 30:379-386 (1993)). In someembodiments, several linkers may be included in order to take advantageof desired properties of each linker.

Chemical linkers and peptide linkers may be inserted by covalentlycoupling the linker to the domain of MTSP protein and the targetingagent. The heterobifunctional agents, described below, may be used toeffect such covalent coupling. Peptide linkers may also be linked byexpressing DNA encoding the linker and TA, linker and targeted agent, orlinker, targeted agent and TA as a fusion protein. Flexible linkers andlinkers that increase solubility of the conjugates are contemplated foruse, either alone or with other linkers are also contemplated herein.

a) Acid Cleavable, Photocleavable and Heat Sensitive Linkers

Acid cleavable linkers, photocleavable and heat sensitive linkers mayalso be used, particularly where it may be necessary to cleave thedomain of MTSP protein to permit it to be more readily accessible toreaction. Acid cleavable linkers include, but are not limited to,bismaleimideothoxy propane; and adipic acid dihydrazide linkers (see,e.g., Fattom et al. (1992) Infection & Immun. 60:584-589) and acidlabile transferrin conjugates that contain a sufficient portion oftransferrin to permit entry into the intracellular transferrin cyclingpathway (see, e.g., Welhöner et al. (1991) J. Biol. Chem.266:4309-4314).

Photocleavable linkers are linkers that are cleaved upon exposure tolight (see, e.g., Goldmacher et al. (1992) Bioconj. Chem. 3:104-107,which linkers are herein incorporated by reference), thereby releasingthe targeted agent upon exposure to light. Photocleavable linkers thatare cleaved upon exposure to light are known (see, e.g., Hazum et al.(1981) in Pept., Proc. Eur. Pept. Symp., 16th, Brunfeldt, K (Ed), pp.105-110, which describes the use of a nitrobenzyl group as aphotocleavable protective group for cysteine; Yen et al. (1989)Makromol. Chem 190:69-82, which describes water soluble photocleavablecopolymers, including hydroxypropylmethacrylamide copolymer, glycinecopolymer, fluorescein copolymer and methylrhodamine copolymer;Goldmacher et al. (1992) Bioconj. Chem. 3:104-107, which describes across-linker and reagent that undergoes photolytic degradation uponexposure to near UV light (350 nm); and Senter et al. (1985) Photochem.Photobiol 42:231-237, which describes nitrobenzyloxycarbonyl chloridecross linking reagents that produce photocleavable linkages), therebyreleasing the targeted agent upon exposure to light. Such linkers wouldhave particular use in treating dermatological or ophthalmic conditionsthat can be exposed to light using fiber optics. After administration ofthe conjugate, the eye or skin or other body part can be exposed tolight, resulting in release of the targeted moiety from the conjugate.Such photocleavable linkers are useful in connection with diagnosticprotocols in which it is desirable to remove the targeting agent topermit rapid clearance from the body of the animal.

b) Other Linkers for Chemical Conjugation

Other linkers, include trityl linkers, particularly, derivatized tritylgroups to generate a genus of conjugates that provide for release oftherapeutic agents at various degrees of acidity or alkalinity. Theflexibility thus afforded by the ability to preselect the pH range atwhich the therapeutic agent will be released allows selection of alinker based on the known physiological differences between tissues inneed of delivery of a therapeutic agent (see, e.g., U.S. Pat. No.5,612,474). For example, the acidity of tumor tissues appears to belower than that of normal tissues.

c) Peptide Linkers

The linker moieties can be peptides. Peptide linkers can be employed infusion proteins and also in chemically linked conjugates. The peptidetypically has from about 2 to about 60 amino acid residues, for examplefrom about 5 to about 40, or from about 10 to about 30 amino acidresidues. The length selected will depend upon factors, such as the usefor which the linker is included.

Peptide linkers are advantageous when the targeting agent isproteinaceous. For example, the linker moiety can be a flexible spaceramino acid sequence, such as those known in single-chain antibodyresearch. Examples of such known linker moieties include, but are notlimited to, peptides, such as (Gly_(m)Ser)_(n) and (Ser_(m)Gly)_(n), inwhich n is 1 to 6, preferably 1 to 4, more preferably 2 to 4, and m is 1to 6, preferably 1 to 4, more preferably 2 to 4, enzyme cleavablelinkers and others.

Additional linking moieties are described, for example, in Huston etal., Proc. Natl. Acad. Sci. U.S.A. 85:5879-5883, 1988; Whitlow, M., etal., Protein Engineering 6:989-995, 1993; Newton et al., Biochemistry35:545-553, 1996; A. J. Cumber et al., Bioconj. Chem. 3:397-401, 1992;Ladumer et al., J. Mol. Biol. 273:330-337, 1997; and U.S. Pat. No.4,894,443. In some embodiments, several linkers may be included in orderto take advantage of desired properties of each linker.

3. Targeting Agents

Any agent that facilitates detection, immobilization, or purification ofthe conjugate is contemplated for use herein. For chemical conjugatesany moiety that has such properties is contemplated; for fusionproteins, the targeting agent is a protein, peptide or fragment thereofthat sufficient to effects the targeting activity. Preferred targetingagents are those that deliver the MTSP protein or portion thereof toselected cells and tissues. Such agents include tumor specificmonoclonal antibodies and portions thereof, growth factors, such as FGF,EGF, PDGF, VEGF, cytokines, including chemokines, and other such agents.

4. Nucleic Acids, Plasmids and Cells

Isolated nucleic acid fragments encoding fusion proteins are provided.The nucleic acid fragment that encodes the fusion protein includes: a)nucleic acid encoding a protease domain of an MTSP protein encoded by anucleic acid that hybridizes to a nucleic acid having the nucleotidesequence set forth in the SEQ. ID NO:1; and b) nucleic acid encoding aprotein, peptide or effective fragment thereof that facilitates: i)affinity isolation or purification of the fusion protein; ii) attachmentof the fusion protein to a surface; or iii) detection of the fusionprotein. Preferably, the nucleic acid is DNA.

Plasmids for replication and vectors for expression that contain theabove nucleic acid fragments are also provided. Cells containing theplasmids and vectors are also provided. The cells can be any suitablehost including, but are not limited to, bacterial cells, yeast cells,fungal cells, plant cells, insect cell and animal cells. The nucleicacids, plasmids, and cells containing the plasmids can be preparedaccording to methods known in the art including any described herein.

Also provided are methods for producing the above fusion proteins. Anexemplary method includes the steps of growing, i.e. culturing the cellsso that the proliferate, cells containing a plasmid encoding the fusionprotein under conditions whereby the fusion protein is expressed by thecell, and recovering the expressed fusion protein. Methods forexpressing and recovering recombinant proteins are well known in the art(See generally, Current Protocols in Molecular Biology (1998) §16, JohnWiley & Sons, Inc.) and such methods can be used for expressing andrecovering the expressed fusion proteins. Preferably, the recombinantexpression and recovery methods disclosed in Section B can be used.

The recovered fusion proteins can be isolated or purified by methodsknown in the art such as centrifugation, filtration, chromatograph,electrophoresis, immunoprecipitation, etc., or by a combination thereof(See generally, Current Protocols in Molecular Biology (1998) §10, JohnWiley & Sons, Inc.). Preferably, the recovered fusion protein isisolated or purified through affinity binding between the protein orpeptide fragment of the fusion protein and an affinity binding moiety.As discussed in the above sections regarding the construction of thefusion proteins, any affinity binding pairs can be constructed and usedin the isolation or purification of the fusion proteins. For example,the affinity binding pairs can be protein binding sequences/protein, DNAbinding sequences/DNA sequences, RNA binding sequences/RNA sequences,lipid binding sequences/lipid, polysaccharide bindingsequences/polysaccharide, or metal binding sequences/metal.

5. Immobilization and Supports or Substrates Therefor

In certain embodiments, where the targeting agents are designed forlinkage to surfaces, the MTSP protein can be attached by linkage such asionic or covalent, non-covalent or other chemical interaction, to asurface of a support or matrix material. Immobilization may be effecteddirectly or via a linker. The MTSP protein may be immobilized on anysuitable support, including, but are not limited to, silicon chips, andother supports described herein and known to those of skill in the art.A plurality of MTSP protein or protease domains thereof may be attachedto a support, such as an array (i.e., a pattern of two or more) ofconjugates on the surface of a silicon chip or other chip for use inhigh throughput protocols and formats.

It is also noted that the domains of the MTSP protein can be linkeddirectly to the surface or via a linker without a targeting agent linkedthereto. Hence chips containing arrays of the domains of the MTSPprotein.

The matrix material or solid supports contemplated herein are generallyany of the insoluble materials known to those of skill in the art toimmobilize ligands and other molecules, and are those that used in manychemical syntheses and separations. Such supports are used, for example,in affinity chromatography, in the immobilization of biologically activematerials, and during chemical syntheses of biomolecules, includingproteins, amino acids and other organic molecules and polymers. Thepreparation of and use of supports is well known to those of skill inthis art; there are many such materials and preparations thereof known.For example, naturally-occurring support materials, such as agarose andcellulose, may be isolated from their respective sources, and processedaccording to known protocols, and synthetic materials may be prepared inaccord with known protocols.

The supports are typically insoluble materials that are solid, porous,deformable, or hard, and have any required structure and geometry,including, but not limited to: beads, pellets, disks, capillaries,hollow fibers, needles, solid fibers, random shapes, thin films andmembranes. Thus, the item may be fabricated from the matrix material orcombined with it, such as by coating all or part of the surface orimpregnating particles.

Typically, when the matrix is particulate, the particles are at leastabout 10-2000 μM, but may be smaller or larger, depending upon theselected application. Selection of the matrices will be governed, atleast in part, by their physical and chemical properties, such assolubility, functional groups, mechanical stability, surface areaswelling propensity, hydrophobic or hydrophilic properties and intendeduse.

If necessary, the support matrix material can be treated to contain anappropriate reactive moiety. In some cases, the support matrix materialalready containing the reactive moiety may be obtained commercially. Thesupport matrix material containing the reactive moiety may thereby serveas the matrix support upon which molecules are linked. Materialscontaining reactive surface moieties such as amino silane linkages,hydroxyl linkages or carboxysilane linkages may be produced by wellestablished surface chemistry techniques involving silanizationreactions, or the like. Examples of these materials are those havingsurface silicon oxide moieties, covalently linked togamma-aminopropylsilane, and other organic moieties;N-[3-(triethyoxysilyl)propyl]phthelamic acid; andbis-(2-hydroxyethyl)aminopropyltriethoxysilane. Exemplary of readilyavailable materials containing amino group reactive functionalities,include, but are not limited to, para-aminophenyltriethyoxysilane. Alsoderivatized polystyrenes and other such polymers are well known andreadily available to those of skill in this art (e.g., the Tentagel®Resins are available with a multitude of functional groups, and are soldby Rapp Polymere, Tubingen, Germany; see, U.S. Pat. No. 4,908,405 andU.S. Pat. No. 5,292,814; see, also Butz et al., Peptide Res., 7:20-23(1994); and Kleine et al., Immunobiol., 190:53-66 (1994)).

These matrix materials include any material that can act as a supportmatrix for attachment of the molecules of interest. Such materials areknown to those of skill in this art, and include those that are used asa support matrix. These materials include, but are not limited to,inorganics, natural polymers, and synthetic polymers, including, but arenot limited to: cellulose, cellulose derivatives, acrylic resins, glass,silica gels, polystyrene, gelatin, polyvinyl pyrrolidone, co-polymers ofvinyl and acrylamide, polystyrene cross-linked with divinylbenzene andothers (see, Merrifield, Biochemistry, 3:1385-1390 (1964)),polyacrylamides, latex gels, polystyrene, dextran, polyacrylamides,rubber, silicon, plastics, nitrocellulose, celluloses, natural sponges.Of particular interest herein, are highly porous glasses (see, e.g.,U.S. Pat. No. 4,244,721) and others prepared by mixing a borosilicate,alcohol and water.

Synthetic supports include, but are not limited to: acrylamides,dextran-derivatives and dextran co-polymers, agarose-polyacrylamideblends, other polymers and co-polymers with various functional groups,methacrylate derivatives and co-polymers, polystyrene and polystyrenecopolymers (see, e.g., Merrifield, Biochemistry, 3:1385-1390 (1964);Berg et al., in Innovation Perspect. Solid Phase Synth. Collect. Pap.,Int. Symp., 1st, Epton, Roger (Ed), pp. 453-459 (1990); Berg et al.,Pept., Proc. Eur. Pept. Symp., 20th, Jung, G. et al. (Eds), pp. 196-198(1989); Berg et al., J. Am. Chem. Soc., 111:8024-8026 (1989); Kent etal., Isr. J. Chem., 17:243-247 (1979); Kent et al., J. Org. Chem.,43:2845-2852 (1978); Mitchell et al., Tetrahedron Lett., 42:3795-3798(1976); U.S. Pat. No. 4,507,230; U.S. Pat. No. 4,006,117; and U.S. Pat.No. 5,389,449). Such materials include those made from polymers andco-polymers such as polyvinylalcohols, acrylates and acrylic acids suchas polyethylene-co-acrylic acid, polyethylene-co-methacrylic acid,polyethylene-co-ethylacrylate, polyethylene-co-methyl acrylate,polypropylene-co-acrylic acid, polypropylene-co-methyl-acrylic acid,polypropylene-co-ethylacrylate, polypropylene-co-methyl acrylate,polyethylene-co-vinyl acetate, polypropylene-co-vinyl acetate, and thosecontaining acid anhydride groups such as polyethylene-co-maleicanhydride and polypropylene-co-maleic anhydride. Liposomes have alsobeen used as solid supports for affinity purifications (Powell et al.Biotechnol. Bioeng., 33:173 (1989)).

Numerous methods have been developed for the immobilization of proteinsand other biomolecules onto solid or liquid supports (see, e.g.,Mosbach, Methods in Enzymology, 44 (1976); Weetall, Immobilized Enzymes,Antigens, Antibodies, and Peptides, (1975); Kennedy et al., Solid PhaseBiochemistry, Analytical and Synthetic Aspects, Scouten, ed., pp.253-391 (1983); see, generally, Affinity Techniques. EnzymePurification: Part B. Methods in Enzymology, Vol. 34, ed. W. B. Jakoby,M. Wilchek, Acad. Press, N.Y. (1974); and Immobilized Biochemicals andAffinity Chromatography, Advances in Experimental Medicine and Biology,vol. 42, ed. R. Dunlap, Plenum Press, N.Y. (1974)).

Among the most commonly used methods are absorption and adsorption orcovalent binding to the support, either directly or via a linker, suchas the numerous disulfide linkages, thioether bonds, hindered disulfidebonds, and covalent bonds between free reactive groups, such as amineand thiol groups, known to those of skill in art (see, e.g., the PIERCECATALOG, ImmunoTechnology Catalog & Handbook, 1992-1993, which describesthe preparation of and use of such reagents and provides a commercialsource for such reagents; Wong, Chemistry of Protein Conjugation andCross Linking, CRC Press (1993); see also DeWitt et al., Proc. Natl.Acad. Sci. U.S.A., 90:6909 (1993); Zuckermann et al., J. Am. Chem. Soc.,114:10646 (1992); Kurth et al., J. Am. Chem. Soc., 116:2661 (1994);Ellman et al., Proc. Natl. Acad. Sci. U.S.A., 91:4708 (1994);Sucholeiki, Tetrahedron Lttrs., 35:7307 (1994); Su-Sun Wang, J. Org.Chem., 41:3258 (1976); Padwa et al., J. Org. Chem., 41:3550 (1971); andVedejs et al., J. Org. Chem., 49:575 (1984), which describephotosensitive linkers).

To effect immobilization, a composition containing the protein or otherbiomolecule is contacted with a support material such as alumina,carbon, an ion-exchange resin, cellulose, glass or a ceramic.Fluorocarbon polymers have been used as supports to which biomoleculeshave been attached by adsorption (see, U.S. Pat. No. 3,843,443;Published International PCT Application WO/86 03840).

J. PROGNOSIS AND DIAGNOSIS

MTSP protein proteins, domains, analogs, and derivatives thereof, andencoding nucleic acids (and sequences complementary thereto), andanti-MTSP protein antibodies, can be used in diagnostics. Such moleculescan be used in assays, such as immunoassays, to detect, prognose,diagnose, or monitor various conditions, diseases, and disordersaffecting MTSP protein expression, or monitor the treatment thereof Forpurposes herein, the presence of MTSPs in body fluids or tumor tissuesare of particular interest.

In particular, such an immunoassay is carried out by a method includingcontacting a sample derived from a patient with an anti-MTSP proteinantibody under conditions such that specific binding can occur, anddetecting or measuring the amount of any specific binding by theantibody. In a specific aspect, such binding of antibody, in tissuesections, can be used to detect aberrant MTSP protein localization oraberrant (e.g., low or absent) levels of MTSP protein. In a specificembodiment, antibody to MTSP protein can be used to assay in a patienttissue or serum sample for the presence of MTSP protein where anaberrant level of MTSP protein is an indication of a diseased condition.

The immunoassays which can be used include but are not limited tocompetitive and non-competitive assay systems using techniques such aswestern blots, radioimmunoassays, ELISA (enzyme linked immunosorbentassay), “sandwich” immunoassays, immunoprecipitation assays, precipitinreactions, gel diffusion precipitin reactions, immunodiffusion assays,agglutination assays, complement-fixation assays, immunoradiometricassays, fluorescent immunoassays, protein A immunoassays, to name but afew.

MTSP protein genes and related nucleic acid sequences and subsequences,including complementary sequences, can also be used in hybridizationassays. MTSP protein nucleic acid sequences, or subsequences thereofcontaining about at least 8 nucleotides, preferably 14 or 16 or 30 ormore continugous nucleotides, can be used as hybridization probes.Hybridization assays can be used to detect, prognose, diagnose, ormonitor conditions, disorders, or disease states associated withaberrant changes in MTSP protein expression and/or activity as describedherein. In particular, such a hybridization assay is carried out by amethod by contacting a sample containing nucleic acid with a nucleicacid probe capable of hybridizing to MTSP protein encoding DNA or RNA,under conditions such that hybridization can occur, and detecting ormeasuring any resulting hybridization.

In a specific embodiment, a method of diagnosing a disease or disordercharacterized by detecting an aberrant level of an MTSP protein in asubject is provided herein by measuring the level of the DNA, RNA,protein or functional activity of the epithelial MTSP protein at leastpartially encoded by a nucleic acid that hybridizes to a nucleic acidhaving the nucleotide sequence set forth in the SEQ. ID NO:1 in a samplederived from the subject, wherein an increase or decrease in the levelof the DNA, RNA, protein or functional activity of the MTSP protein,relative to the level of the DNA, RNA, protein or functional activityfound in an analogous sample not having the disease or disorderindicates the presence of the disease or disorder in the subject.

Kits for diagnostic use are also provided, that contain in one or morecontainers an anti-MTSP protein antibody, particularly anti-MTSP3 oranti=MTSP4, and, optionally, a labeled binding partner to the antibody.Alternatively, the anti-MTSP protein antibody can be labeled (with adetectable marker, e.g., a chemiluminescent, enzymatic, fluorescent, orradioactive moiety). A kit is also provided that includes in one or morecontainers a nucleic acid probe capable of hybridizing to MTSPprotein-encoding RNA. In a specific embodiment, a kit can comprise inone or more containers a pair of primers (e.g., each in the size rangeof 6-30 nucleotides) that are capable of priming amplification [e.g., bypolymerase chain reaction (see e.g., Innis et al., 1990, PCR Protocols,Academic Press, Inc., San Diego, Calif.), ligase chain reaction (see EP320,308) use of Qβ replicase, cyclic probe reaction, or other methodsknown in the art under appropriate reaction conditions of at least aportion of an MTSP protein-encoding nucleic acid. A kit can optionallyfurther comprise in a container a predetermined amount of a purifiedMTSP protein or nucleic acid, e.g., for use as a standard or control.

K. PHARMACEUTICAL COMPOSITIONS AND MODES OF ADMINISTRATION

1. Components of the Compositions

Pharmaceutical compositions containing the identified compounds thatmodulate the activity of an MTSP protein are provided herein. Alsoprovided are combinations of a compound that modulates the activity ofan MTSP protein and another treatment or compound for treatment of aneoplastic disorder, such as a chemotherapeutic compound.

The MTSP protein modulator and the anti-tumor agent can be packaged asseparate compositions for administration together or sequentially orintermittently. Alternatively, they can provided as a single compositionfor administration or as two compositions for administration as a singlecomposition. The combinations can be packaged as kits.

a. MTSP Protein Inhibitors

Any MTSP protein inhibitors, including those described herein when usedalone or in combination with other compounds, that can alleviate,reduce, ameliorate, prevent, or place or maintain in a state ofremission of clinical symptoms or diagnostic markers associated withneoplastic diseases, including undesired and/or uncontrolledangiogenesis, can be used in the present combinations.

In one embodiment, the MTSP protein inhibitor is an antibody or fragmentthereof that specifically reacts with an MTSP protein or the proteasedomain thereof, an inhibitor of the MTSP protein production, aninhibitor of the epithelial MTSP protein membrane-localization, or anyinhibitor of the expression of or, especially, the activity of an MTSPprotein.

b. Anti-Angiogenic Agents and Anti-Tumor Agents

Any anti-angiogenic agents and anti-tumor agents, including thosedescribed herein, when used alone or in combination with othercompounds, that can alleviate, reduce, ameliorate, prevent, or place ormaintain in a state of remission of clinical symptoms or diagnosticmarkers associated with undesired and/or uncontrolled angiogenesisand/or tumor growth and metastasis, particularly solid neoplasms,vascular malformations and cardiovascular disorders, chronicinflammatory diseases and aberrant wound repairs, circulatory disorders,crest syndromes, dermatological disorders, or ocular disorders, can beused in the combinations. Also contemplated are anti-tumor agents foruse in combination with an inhibitor of an MTSP protein.

c. Anti-Tumor Agents and Anti-Angiogenic Agents

The compounds identified by the methods provided herein or providedherein can be used in combination with anti-tumor agents and/oranti-angiogenesis agents.

2. Formulations and Route of Administration

The compounds herein and agents are preferably formulated aspharmaceutical compositions, preferably for single dosageadministration. The concentrations of the compounds in the formulationsare effective for delivery of an amount, upon administration, that iseffective for the intended treatment. Typically, the compositions areformulated for single dosage administration. To formulate a composition,the weight fraction of a compound or mixture thereof is dissolved,suspended, dispersed or otherwise mixed in a selected vehicle at aneffective concentration such that the treated condition is relieved orameliorated. Pharmaceutical carriers or vehicles suitable foradministration of the compounds provided herein include any suchcarriers known to those skilled in the art to be suitable for theparticular mode of administration.

In addition, the compounds may be formulated as the solepharmaceutically active ingredient in the composition or may be combinedwith other active ingredients. Liposomal suspensions, includingtissue-targeted liposomes, may also be suitable as pharmaceuticallyacceptable carriers. These may be prepared according to methods known tothose skilled in the art. For example, liposome formulations may beprepared as described in U.S. Pat. No. 4,522,811.

The active compound is included in the pharmaceutically acceptablecarrier in an amount sufficient to exert a therapeutically useful effectin the absence of undesirable side effects on the patient treated. Thetherapeutically effective concentration may be determined empirically bytesting the compounds in known in vitro and in vivo systems, such as theassays provided herein.

The concentration of active compound in the drug composition will dependon absorption, inactivation and excretion rates of the active compound,the physicochemical characteristics of the compound, the dosageschedule, and amount administered as well as other factors known tothose of skill in the art.

Typically a therapeutically effective dosage is contemplated. Theamounts administered may be on the order of 0.001 to 1 mg/ml, preferablyabout 0.005-0.05 mg/ml, more preferably about 0.01 mg/ml, of bloodvolume. Pharmaceutical dosage unit forms are prepared to provide fromabout 1 mg to about 1000 mg and preferably from about 10 to about 500mg, more preferably about 25-75 mg of the essential active ingredient ora combination of essential ingredients per dosage unit form. The precisedosage can be empirically determined.

The active ingredient may be administered at once, or may be dividedinto a number of smaller doses to be administered at intervals of time.It is understood that the precise dosage and duration of treatment is afunction of the disease being treated and may be determined empiricallyusing known testing protocols or by extrapolation from in vivo or invitro test data. It is to be noted that concentrations and dosage valuesmay also vary with the severity of the condition to be alleviated. It isto be further understood that for any particular subject, specificdosage regimens should be adjusted over time according to the individualneed and the professional judgment of the person administering orsupervising the administration of the compositions, and that theconcentration ranges set forth herein are exemplary only and are notintended to limit the scope or use of the claimed compositions andcombinations containing them.

Preferred pharmaceutically acceptable derivatives include acids, salts,esters, hydrates, solvates and prodrug forms. The derivative istypically selected such that its pharmacokinetic properties are superiorto the corresponding neutral compound.

Thus, effective concentrations or amounts of one or more of thecompounds provided herein or pharmaceutically acceptable derivativesthereof are mixed with a suitable pharmaceutical carrier or vehicle forsystemic, topical or local administration to form pharmaceuticalcompositions. Compounds are included in an amount effective forameliorating or treating the disorder for which treatment iscontemplated. The concentration of active compound in the compositionwill depend on absorption, inactivation, excretion rates of the activecompound, the dosage schedule, amount administered, particularformulation as well as other factors known to those of skill in the art.

Solutions or suspensions used for parenteral, intradermal, subcutaneous,or topical application can include any of the following components: asterile diluent, such as water for injection, saline solution, fixedoil, polyethylene glycol, glycerine, propylene glycol or other syntheticsolvent; antimicrobial agents, such as benzyl alcohol and methylparabens; antioxidants, such as ascorbic acid and sodium bisulfite;chelating agents, such as ethylenediaminetetraacetic acid (EDTA);buffers, such as acetates, citrates and phosphates; and agents for theadjustment of tonicity such as sodium chloride or dextrose. Parenteralpreparations can be enclosed in ampules, disposable syringes or singleor multiple dose vials made of glass, plastic or other suitablematerial.

In instances in which the compounds exhibit insufficient solubility,methods for solubilizing compounds may be used. Such methods are knownto those of skill in this art, and include, but are not limited to,using cosolvents, such as dimethylsulfoxide (DMSO), using surfactants,such as Tween®, or dissolution in aqueous sodium bicarbonate.Derivatives of the compounds, such as prodrugs of the compounds may alsobe used in formulating effective pharmaceutical compositions. Forophthalmic indications, the compositions are formulated in anophthalmically acceptable carrier. For the ophthalmic uses herein, localadministration, either by topical administration or by injection ispreferred. Time release formulations are also desirable. Typically, thecompositions are formulated for single dosage administration, so that asingle dose administers an effective amount.

Upon mixing or addition of the compound with the vehicle, the resultingmixture may be a solution, suspension, emulsion or other composition.The form of the resulting mixture depends upon a number of factors,including the intended mode of administration and the solubility of thecompound in the selected carrier or vehicle. If necessary,pharmaceutically acceptable salts or other derivatives of the compoundsare prepared.

The compound is included in the pharmaceutically acceptable carrier inan amount sufficient to exert a therapeutically useful effect in theabsence of undesirable side effects on the patient treated. It isunderstood that number and degree of side effects depends upon thecondition for which the compounds are administered. For example, certaintoxic and undesirable side effects are tolerated when treatinglife-threatening illnesses that would not be tolerated when treatingdisorders of lesser consequence.

The compounds can also be mixed with other active materials, that do notimpair the desired action, or with materials that supplement the desiredaction known to those of skill in the art. The formulations of thecompounds and agents for use herein include those suitable for oral,rectal, topical, inhalational, buccal (e.g., sublingual), parenteral(e.g., subcutaneous, intramuscular, intradermal, or intravenous),transdermal administration or any route. The most suitable route in anygiven case will depend on the nature and severity of the condition beingtreated and on the nature of the particular active compound which isbeing used. The formulations are provided for administration to humansand animals in unit dosage forms, such as tablets, capsules, pills,powders, granules, sterile parenteral solutions or suspensions, and oralsolutions or suspensions, and oil-water emulsions containing suitablequantities of the compounds or pharmaceutically acceptable derivativesthereof. The pharmaceutically therapeutically active compounds andderivatives thereof are typically formulated and administered inunit-dosage forms or multiple-dosage forms. Unit-dose forms as usedherein refers to physically discrete units suitable for human and animalsubjects and packaged individually as is known in the art. Eachunit-dose contains a predetermined quantity of the therapeuticallyactive compound sufficient to produce the desired therapeutic effect, inassociation with the required pharmaceutical carrier, vehicle ordiluent. Examples of unit-dose forms include ampoules and syringes andindividually packaged tablets or capsules. Unit-dose forms may beadministered in fractions or multiples thereof. A multiple-dose form isa plurality of identical unit-dosage forms packaged in a singlecontainer to be administered in segregated unit-dose form. Examples ofmultiple-dose forms include vials, bottles of tablets or capsules orbottles of pints or gallons. Hence, multiple dose form is a multiple ofunit-doses which are not segregated in packaging.

The composition can contain along with the active ingredient: a diluentsuch as lactose, sucrose, dicalcium phosphate, orcarboxymethylcellulose; a lubricant, such as magnesium stearate, calciumstearate and talc; and a binder such as starch, natural gums, such asgum acaciagelatin, glucose, molasses, polvinylpyrrolidine, cellulosesand derivatives thereof, povidone, crospovidones and other such bindersknown to those of skill in the art. Liquid pharmaceuticallyadministrable compositions can, for example, be prepared by dissolving,dispersing, or otherwise mixing an active compound as defined above andoptional pharmaceutical adjuvants in a carrier, such as, for example,water, saline, aqueous dextrose, glycerol, glycols, ethanol, and thelike, to thereby form a solution or suspension. If desired, thepharmaceutical composition to be administered may also contain minoramounts of nontoxic auxiliary substances such as wetting agents,emulsifying agents, or solubilizing agents, pH buffering agents and thelike, for example, acetate, sodium citrate, cyclodextrine derivatives,sorbitan monolaurate, triethanolamine sodium acetate, triethanolamineoleate, and other such agents. Methods of preparing such dosage formsare known, or will be apparent, to those skilled in this art (see, e.g.,Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton,Pa., 15th Edition, 1975). The composition or formulation to beadministered will contain a quantity of the active compound in an amountsufficient to alleviate the symptoms of the treated subject.

Dosage forms or compositions containing active ingredient in the rangeof 0.005% to 100% with the balance made up from non-toxic carrier may beprepared. For oral administration, the pharmaceutical compositions maytake the form of, for example, tablets or capsules prepared byconventional means with pharmaceutically acceptable excipients such asbinding agents (e.g., pregelatinized maize starch, polyvinyl pyrrolidoneor hydroxypropyl methylcellulose); fillers (e.g., lactose,microcrystalline cellulose or calcium hydrogen phosphate); lubricants(e.g., magnesium stearate, talc or silica); disintegrants (e.g., potatostarch or sodium starch glycolate); or wetting agents (e.g., sodiumlauryl sulphate). The tablets may be coated by methods well-known in theart.

The pharmaceutical preparation may also be in liquid form, for example,solutions, syrups or suspensions, or may be presented as a drug productfor reconstitution with water or other suitable vehicle before use. Suchliquid preparations may be prepared by conventional means withpharmaceutically acceptable additives such as suspending agents (e.g.,sorbitol syrup, cellulose derivatives or hydrogenated edible fats);emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles(e.g., almond oil, oily esters, or fractionated vegetable oils); andpreservatives (e.g., methyl or propyl-p-hydroxybenzoates or sorbicacid).

Formulations suitable for rectal administration are preferably presentedas unit dose suppositories. These may be prepared by admixing the activecompound with one or more conventional solid carriers, for example,cocoa butter, and then shaping the resulting mixture.

Formulations suitable for topical application to the skin or to the eyepreferably take the form of an ointment, cream, lotion, paste, gel,spray, aerosol and oil. Carriers which may be used include vaseline,lanoline, polyethylene glycols, alcohols, and combinations of two ormore thereof. The topical formulations may further advantageouslycontain 0.05 to 15 percent by weight of thickeners selected from amonghydroxypropyl methyl cellulose, methyl cellulose, polyvinylpyrrolidone,polyvinyl alcohol, poly(alkylene glycols), poly/hydroxyalkyl,(meth)acrylates or poly(meth)acrylamides. A topical formulation is oftenapplied by instillation or as an ointment into the conjunctival sac. Itcan also be used for irrigation or lubrication of the eye, facialsinuses, and external auditory meatus. It may also be injected into theanterior eye chamber and other places. The topical formulations in theliquid state may be also present in a hydrophilic three-dimensionalpolymer matrix in the form of a strip, contact lens, and the like fromwhich the active components are released.

For administration by inhalation, the compounds for use herein can bedelivered in the form of an aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant, e.g.,dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane, carbon dioxide or other suitable gas. In thecase of a pressurized aerosol, the dosage unit may be determined byproviding a valve to deliver a metered amount. Capsules and cartridgesof, e.g., gelatin, for use in an inhaler or insufflator may beformulated containing a powder mix of the compound and a suitable powderbase such as lactose or starch.

Formulations suitable for buccal (sublingual) administration include,for example, lozenges containing the active compound in a flavored base,usually sucrose and acacia or tragacanth; and pastilles containing thecompound in an inert base such as gelatin and glycerin or sucrose andacacia.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampules orin multi-dose containers, with an added preservative. The compositionsmay be suspensions, solutions or emulsions in oily or aqueous vehicles,and may contain formulatory agents such as suspending, stabilizingand/or dispersing agents. Alternatively, the active ingredient may be inpowder form for reconstitution with a suitable vehicle, e.g., sterilepyrogen-free water or other solvents, before use.

Formulations suitable for transdermal administration may be presented asdiscrete patches adapted to remain in intimate contact with theepidermis of the recipient for a prolonged period of time. Such patchessuitably contain the active compound as an optionally buffered aqueoussolution of, for example, 0.1 to 0.2 M concentration with respect to theactive compound. Formulations suitable for transdermal administrationmay also be delivered by iontophoresis (see, e.g., PharmaceuticalResearch 3(6), 318 (1986)) and typically take the form of an optionallybuffered aqueous solution of the active compound.

The pharmaceutical compositions may also be administered by controlledrelease means and/or delivery devices (see, e.g., in U.S. Pat. Nos.3,536,809; 3,598,123; 3,630,200; 3,845,770; 3,847,770; 3,916,899;4,008,719; 4,687,610; 4,769,027; 5,059,595; 5,073,543; 5,120,548;5,354,566; 5,591,767; 5,639,476; 5,674,533 and 5,733,566).

Desirable blood levels may be maintained by a continuous infusion of theactive agent as ascertained by plasma levels. It should be noted thatthe attending physician would know how to and when to terminate,interrupt or adjust therapy to lower dosage due to toxicity, or bonemarrow, liver or kidney dysfunctions. Conversely, the attendingphysician would also know how to and when to adjust treatment to higherlevels if the clinical response is not adequate (precluding toxic sideeffects).

The efficacy and/or toxicity of the MTSP protein inhibitor(s), alone orin combination with other agents can also be assessed by the methodsknown in the art (See generally, O'Reilly, Investigational New Drugs,15:5-13 (1997)).

The active compounds or pharmaceutically acceptable derivatives may beprepared with carriers that protect the compound against rapidelimination from the body, such as time release formulations orcoatings.

Kits containing the compositions and/or the combinations withinstructions for administration thereof are provided. The kit mayfurther include a needle or syringe, preferably packaged in sterileform, for injecting the complex, and/or a packaged alcohol pad.Instructions are optionally included for administration of the activeagent by a clinician or by the patient.

Finally, the compounds or MTSP proteins or protease domains thereof orcompositions containing any of the preceding agents may be packaged asarticles of manufacture containing packaging material, a compound orsuitable derivative thereof provided herein, which is effective fortreatment of a diseases or disorders contemplated herein, within thepackaging material, and a label that indicates that the compound or asuitable derivative thereof is for treating the diseases or disorderscontemplated herein. The label can optionally include the disorders forwhich the therapy is warranted.

L. METHODS OF TREATMENT

The compounds identified by the methods herein are used for treating orpreventing neoplastic diseases in an animal, particularly a mammal,including a human, is provided herein. In one embodiment, the methodincludes administering to a mammal an effective amount of an inhibitorof an MTSP protein, whereby the disease or disorder is treated orprevented. In a preferred embodiment, the MTSP protein inhibitor used inthe treatment or prevention is administered with a pharmaceuticallyacceptable carrier or excipient. The mammal treated can be a human.

The inhibitors provided herein are those identified by the screeningassays. In addition, antibodies and antisense nucleic acids arecontemplated.

The treatment or prevention method can further include administering ananti-angiogenic treatment or agent or anti-tumor agent simultaneouslywith, prior to or subsequent to the MTSP protein inhibitor, which can beany compound identified that inhibits the activity of an MTSP protein,and includes an antibody or a fragment or derivative thereof containingthe binding region thereof against the MTSP protein, an antisensenucleic acid encoding the MTSP protein, and a nucleic acid containing atleast a portion of a gene encoding the MTSP protein into which aheterologous nucleotide sequence has been inserted such that theheterologous sequence inactivates the biological activity of at least aportion of the gene encoding the MTSP protein, in which the portion ofthe gene encoding the MTSP protein flanks the heterologous sequence soas to promote homologous recombination with a genomic gene encoding theMTSP protein.

1. Antisense Treatment

In a specific embodiment, as described hereinabove, MTSP proteinfunction is reduced or inhibited by MTSP protein antisense nucleicacids, to treat or prevent neoplastic disease. The therapeutic orprophylactic use of nucleic acids of at least six nucleotides that areantisense to a gene or cDNA encoding MTSP protein or a portion thereof.An MTSP protein “antisense” nucleic acid as used herein refers to anucleic acid capable of hybridizing to a portion of an MTSP protein RNA(preferably mRNA) by virtue of some sequence complementarily. Theantisense nucleic acid may be complementary to a coding and/or noncodingregion of an MTSP protein mRNA. Such antisense nucleic acids haveutility as therapeutics that reduce or inhibit MTSP protein function,and can be used in the treatment or prevention of disorders as describedsupra.

The MTSP protein antisense nucleic acids are of at least six nucleotidesand are preferably oligonucleotides (ranging from 6 to about 150nucleotides, or more preferably 6 to 50 nucleotides). In specificaspects, the oligonucleotide is at least 10 nucleotides, at least 15nucleotides, at least 100 nucleotides, or at least 125 nucleotides. Theoligonucleotides can be DNA or RNA or chimeric mixtures or derivativesor modified versions thereof, single-stranded or double-stranded. Theoligonucleotide can be modified at the base moiety, sugar moiety, orphosphate backbone. The oligonucleotide may include other appendinggroups such as peptides, or agents facilitating transport across thecell membrane (see, e.g., Letsinger et al., Proc. Natl. Acad. Sci.U.S.A. 86:6553-6556 (1989); Lemaitre et al., Proc. Natl. Acad. Sci.U.S.A. 84:648-652 (1987); PCT Publication No. WO 88/09810, publishedDec. 15, 1988) or blood-brain barrier (see, e.g., PCT Publication No. WO89/10134, published Apr. 25, 1988), hybridization-triggered cleavageagents (see, e.g., Van Der Krol et al., BioTechniques 6:958-976 (1988))or intercalating agents (see, e.g., Zon, Pharm. Res. 5:539-549 (1988)).

The MTSP protein antisense nucleic acid is preferably anoligonucleotide, more preferably of single-stranded DNA. In a preferredaspect, the oligonucleotide includes a sequence antisense to a portionof human MTSP protein. The oligonucleotide may be modified at anyposition on its structure with substituents generally known in the art.

The MTSP protein antisense oligonucleotide may comprise at least onemodified base moiety which is selected from the group including, but notlimited to 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,hypoxanthine, xanthine, 4-acetylcytosine,5-(carboxyhydroxylmethyl)uracil,5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylaminomethyluracil, dihydrouracil,beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine,7-methylguanine, 5-methylaminomethyluracil,5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,5′-methoxycarboxymethyluracil, 5-methoxyuracil,2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),wybutoxosine, pseudouracil, queosine, 2-thiocytosine,5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,uracil-5-oxyacetic acid methylester, uracil-5-oxyacetic acid (v),5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w,and 2,6-diaminopurine.

In another embodiment, the oligonucleotide includes at least onemodified sugar moiety selected from the group including but not limitedto arabinose, 2-fluoroarabinose, xylulose, and hexose. Theoligonucleotide can include at least one modified phosphate backboneselected from a phosphorothioate, a phosphorodithioate, aphosphoramidothioate, a phosphoramidate, a phosphordiamidate, amethylphosphonate, an alkyl phosphotriester, and a formacetal or analogthereof.

The oligonucleotide can be an α-anomeric oligonucleotide. An α-anomericoligonucleotide forms specific double-stranded hybrids withcomplementary RNA in which the strands run parallel to each other(Gautier et al., Nucl. Acids Res. 15:6625-6641 (1987)).

The oligonucleotide may be conjugated to another molecule, e.g., apeptide, hybridization triggered cross-linking agent, transport agentand hybridization-triggered cleavage agent.

The oligonucleotides may be synthesized by standard methods known in theart, e.g. by use of an automated DNA synthesizer (such as arecommercially available from Biosearch, Applied Biosystems, etc.). Asexamples, phosphorothioate oligonucleotides may be synthesized by themethod of Stein et al. (Nucl. Acids Res. 16:3209 (1988)),methylphosphonate oligonucleotides can be prepared by use of controlledpore glass polymer supports (Sarin et al., Proc. Natl. Acad. Sci. U.S.A.85:7448-7451 (1988)), etc.

In a specific embodiment, the MTSP protein antisense oligonucleotideincludes catalytic RNA, or a ribozyme (see, e.g., PCT InternationalPublication WO 90/11364, published Oct. 4, 1990; Sarver et al., Science247:1222-1225 (1990)). In another embodiment, the oligonucleotide is a2′-O-methylribonucleotide (Inoue et al., Nucl. Acids Res. 15:6131-6148(1987)), or a chimeric RNA-DNA analogue (Inoue et al., FEBS Lett.215:327-330 (1987)).

In an alternative embodiment, the MTSP protein antisense nucleic acid isproduced intracellularly by transcription from an exogenous sequence.For example, a vector can be introduced in vivo such that it is taken upby a cell, within which cell the vector or a portion thereof istranscribed, producing an antisense nucleic acid (RNA). Such a vectorwould contain a sequence encoding the MTSP protein antisense nucleicacid. Such a vector can remain episomal or become chromosomallyintegrated, as long as it can be transcribed to produce the desiredantisense RNA. Such vectors can be constructed by recombinant DNAtechnology methods standard in the art. Vectors can be plasmid, viral,or others known in the art, used for replication and expression inmammalian cells. Expression of the sequence encoding the MTSP proteinantisense RNA can be by any promoter known in the art to act inmammalian, preferably human, cells. Such promoters can be inducible orconstitutive. Such promoters include but are not limited to: the SV40early promoter region (Bernoist and Chambon, Nature 290:304-310 (1981),the promoter contained in the 3′ long terminal repeat of Rous sarcomavirus (Yamamoto et al., Cell 22:787-797 (1980), the herpes thymidinekinase promoter (Wagner et al., Proc. Natl. Acad. Sci. U.S.A.78:1441-1445 (1981), the regulatory sequences of the metallothioneingene (Brinster et al., Nature 296:39-42 (1982), etc.

The antisense nucleic acids include sequence complementary to at least aportion of an RNA transcript of an MTSP protein gene, preferably a humanMTSP protein gene. Absolute complementarily, although preferred, is notrequired.

The amount of MTSP protein antisense nucleic acid that will be effectivein the treatment or prevention of neoplastic disease will depend on thenature of the disease, and can be determined empirically by standardclinical techniques. Where possible, it is desirable to determine theantisense cytotoxicity in cells in vitro, and then in useful animalmodel systems prior to testing and use in humans.

2. Gene Therapy

In an exemplary embodiment, nucleic acids that include a sequence ofnucleotides encoding an MTSP protein or functional domains or derivativethereof, are administered to promote MTSP protein function, by way ofgene therapy. Gene therapy refers to therapy performed by theadministration of a nucleic acid to a subject. In this embodiment, thenucleic acid produces its encoded protein that mediates a therapeuticeffect by promoting MTSP protein function. Any of the methods for genetherapy available in the art can be used (see, Goldspiel et al.,Clinical Pharmacy 12:488-505 (1993); Wu and Wu, Biotherapy 3:87-95(1991); Tolstoshev, An. Rev. Pharmacol. Toxicol. 32:573-596 (1993);Mulligan, Science 260:926-932 (1993); and Morgan and Anderson, An. Rev.Biochem. 62:191-217 (1993); TIBTECH 11(5):155-215 (1993). For example,one therapeutic composition for gene therapy includes an MTSPprotein-encoding nucleic acid that is part of an expression vector thatexpresses an MTSP protein or domain, fragment or chimeric proteinthereof in a suitable host. In particular, such a nucleic acid has apromoter operably linked to the MTSP protein coding region, the promoterbeing inducible or constitutive, and, optionally, tissue-specific. Inanother particular embodiment, a nucleic acid molecule is used in whichthe MTSP protein coding sequences and any other desired sequences areflanked by regions that promote homologous recombination at a desiredsite in the genome, thus providing for intrachromosomal expression ofthe MTSP protein nucleic acid (Koller and Smithies, Proc. Natl. Acad.Sci. USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438(1989)).

Delivery of the nucleic acid into a patient may be either direct, inwhich case the patient is directly exposed to the nucleic acid ornucleic acid-carrying vector, or indirect, in which case, cells arefirst transformed with the nucleic acid in vitro, then transplanted intothe patient. These two approaches are known, respectively, as in vivo orex vivo gene therapy.

In a specific embodiment, the nucleic acid is directly administered invivo, where it is expressed to produce the encoded product. This can beaccomplished by any of numerous methods known in the art, e.g., byconstructing it as part of an appropriate nucleic acid expression vectorand administering it so that it becomes intracellular, e.g., byinfection using a defective or attenuated retroviral or other viralvector (see U.S. Pat. No. 4,980,286), or by direct injection of nakedDNA, or by use of microparticle bombardment (e.g., a gene gun;Biolistic, Dupont), or coating with lipids or cell-surface receptors ortransfecting agents, encapsulation in liposomes, microparticles, ormicrocapsules, or by administering it in linkage to a peptide which isknown to enter the nucleus, by administering it in linkage to a ligandsubject to receptor-mediated endocytosis (see e.g., Wu and Wu, J. Biol.Chem. 262:4429-4432 (1987)) (which can be used to target cell typesspecifically expressing the receptors), etc. In another embodiment, anucleic acid-ligand complex can be formed in which the ligand is afusogenic viral peptide to disrupt endosomes, allowing the nucleic acidto avoid lysosomal degradation. In yet another embodiment, the nucleicacid can be targeted in vivo for cell specific uptake and expression, bytargeting a specific receptor (see, e.g., PCT Publications WO 92/06180dated Apr. 16, 1992 (Wu et al.); WO 92/22635 dated Dec. 23, 1992 (Wilsonet al.); WO92/20316 dated Nov. 26, 1992 (Findeis et al.); WO93/14188dated Jul. 22, 1993 (Clarke et al.), WO 93/20221 dated Oct. 14, 1993(Young)). Alternatively, the nucleic acid can be introducedintracellularly and incorporated within host cell DNA for expression, byhomologous recombination (Koller and Smithies, Proc. Natl. Acad. Sci.USA 86:8932-8935 (1989); Zijlstra et al., Nature 342:435-438 (1989)).

In a specific embodiment, a viral vector that contains the MTSP proteinnucleic acid is used. For example, a retroviral vector can be used (seeMiller et al., Meth. Enzymol. 217:581-599 (1993)). These retroviralvectors have been modified to delete retroviral sequences that are notnecessary for packaging of the viral genome and integration into hostcell DNA. The MTSP protein nucleic acid to be used in gene therapy iscloned into the vector, which facilitates delivery of the gene into apatient. More detail about retroviral vectors can be found in Boesen etal., Biotherapy 6:291-302 (1994), which describes the use of aretroviral vector to deliver the mdr1 gene to hematopoietic stem cellsin order to make the stem cells more resistant to chemotherapy. Otherreferences illustrating the use of retroviral vectors in gene therapyare: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem et al.,Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics andDevel. 3:110-114 (1993).

Adenoviruses are other viral vectors that can be used in gene therapy.Adenoviruses are especially attractive vehicles for delivering genes torespiratory epithelia. Adenoviruses naturally infect respiratoryepithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);and Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993).

Adeno-associated virus (AAV) has also been proposed for use in genetherapy (Walsh et al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993).

Another approach to gene therapy involves transferring a gene to cellsin tissue culture by such methods as electroporation, lipofection,calcium phosphate mediated transfection, or viral infection. Usually,the method of transfer includes the transfer of a selectable marker tothe cells. The cells are then placed under selection to isolate thosecells that have taken up and are expressing the transferred gene. Thosecells are then delivered to a patient.

In this embodiment, the nucleic acid is introduced into a cell prior toadministration in vivo of the resulting recombinant cell. Suchintroduction can be carried out by any method known in the art,including but not limited to transfection, electroporation,microinjection, infection with a viral or bacteriophage vectorcontaining the nucleic acid sequences, cell fusion, chromosome-mediatedgene transfer, microcell-mediated gene transfer, spheroplast fusion,etc. Numerous techniques are known in the art for the introduction offoreign genes into cells (see e.g., Loeffler and Behr, Meth. Enzymol.217:599-618 (1993); Cohen et al., Meth. Enzymol. 217:618-644 (1993);Cline, Pharmac. Ther. 29:69-92 (1985)) and may be used, provided thatthe necessary developmental and physiological functions of the recipientcells are not disrupted. The technique should provide for the stabletransfer of the nucleic acid to the cell, so that the nucleic acid isexpressible by the cell and preferably heritable and expressible by itscell progeny.

The resulting recombinant cells can be delivered to a patient by variousmethods known in the art. In a preferred embodiment, epithelial cellsare injected, e.g., subcutaneously. In another embodiment, recombinantskin cells may be applied as a skin graft onto the patient. Recombinantblood cells (e.g., hematopoietic stem or progenitor cells) arepreferably administered intravenously. The amount of cells envisionedfor use depends on the desired effect, patient state, etc., and can bedetermined by one skilled in the art.

Cells into which a nucleic acid can be introduced for purposes of genetherapy encompass any desired, available cell type, and include but arenot limited to epithelial cells, endothelial cells, keratinocytes,fibroblasts, muscle cells, hepatocytes; blood cells such as Tlymphocytes, B lymphocytes, monocytes, macrophages, neutrophils,eosinophils, megakaryocytes, granulocytes; various stem or progenitorcells, in particular hematopoietic stem or progenitor cells, e.g., asobtained from bone marrow, umbilical cord blood, peripheral blood, fetalliver, etc.

In a preferred embodiment, the cell used for gene therapy is autologousto the patient. In an embodiment in which recombinant cells are used ingene therapy, an MTSP protein nucleic acid is introduced into the cellssuch that it is expressible by the cells or their progeny, and therecombinant cells are then administered in vivo for therapeutic effect.In a specific embodiment, stem or progenitor cells are used. Any stemand/or progenitor cells which can be isolated and maintained in vitrocan potentially be used in accordance with this embodiment. Such stemcells include but are not limited to hematopoietic stem cells (HSC),stem cells of epithelial tissues such as the skin and the lining of thegut, embryonic heart muscle cells, liver stem cells (PCT Publication WO94/08598, dated Apr. 28, 1994), and neural stem cells (Stemple andAnderson, Cell 71:973-985 (1992)).

Epithelial stem cells (ESCs) or keratinocytes can be obtained fromtissues such as the skin and the lining of the gut by known procedures(Rheinwald, Meth. Cell Bio. 21A:229 (1980)). In stratified epithelialtissue such as the skin, renewal occurs by mitosis of stem cells withinthe germinal layer, the layer closest to the basal lamina. Stem cellswithin the lining of the gut provide for a rapid renewal rate of thistissue. ESCs or keratinocytes obtained from the skin or lining of thegut of a patient or donor can be grown in tissue culture (Rheinwald,Meth. Cell Bio. 21A:229 (1980); Pittelkow and Scott, Mayo Clinic Proc.61:771 (1986)). If the ESCs are provided by a donor, a method forsuppression of host versus graft reactivity (e.g., irradiation, drug orantibody administration to promote moderate immunosuppression) can alsobe used.

With respect to hematopoietic stem cells (HSC), any technique whichprovides for the isolation, propagation, and maintenance in vitro of HSCcan be used in this embodiment. Techniques by which this may beaccomplished include (a) the isolation and establishment of HSC culturesfrom bone marrow cells isolated from the future host, or a donor, or (b)the use of previously established long-term HSC cultures, which may beallogeneic or xenogeneic. Non-autologous HSC are used preferably inconjunction with a method of suppressing transplantation immunereactions of the future host/patient. In a particular embodiment, humanbone marrow cells can be obtained from the posterior iliac crest byneedle aspiration (see, e.g., Kodo et al., J. Clin. Invest. 73:1377-1384(1984)). In a preferred embodiment, the HSCs can be made highly enrichedor in substantially pure form. This enrichment can be accomplishedbefore, during, or after long-term culturing, and can be done by anytechniques known in the art. Long-term cultures of bone marrow cells canbe established and maintained by using, for example, modified Dextercell culture techniques (Dexter et al., J. Cell Physiol. 91:335 (1977)or Witlock-Witte culture techniques (Witlock and Witte, Proc. Natl.Acad. Sci. USA 79:3608-3612 (1982)).

In a specific embodiment, the nucleic acid to be introduced for purposesof gene therapy includes an inducible promoter operably linked to thecoding region, such that expression of the nucleic acid is controllableby controlling the presence or absence of the appropriate inducer oftranscription.

3. Prodrugs

A method for treating tumors is provided. The method is practiced byadministering a prodrug that is specifically cleaved by an MTSP torelease an active drug. Upon contact with a cell that expresses MTSPactivity, the prodrug is converted into an active drug. The prodrug canbe a conjugate that contains the active agent, such as an anti-tumordrug, such as a cytotoxic agent, or other atherapeutic agent, linked,linked to a substrate for the targeted MTSP, such that the drug or agentis inactive or unable to enter a cell, in the conjugate, but isactivated upon cleavage. The prodrug, for example, can contain anoligopeptide, preferably a relatively short, less than about 10 aminoacids peptide, that is selectively proteolytically cleaved by thetargeted MTSP. Cytotoxic agents, include, but are not limited to,alkylating agents, antiproliferative agents and tubulin binding agents.Others include, vinca drugs, mitomycins, bleomycins and taxanes.

M. ANIMAL MODELS

Transgenic animal models are provided herein. Such an animal can beinitially produced by promoting homologous recombination between an MTSPprotein gene in its chromosome and an exogenous MTSP protein gene thathas been rendered biologically inactive (preferably by insertion of aheterologous sequence, e.g., an antibiotic resistance gene). In apreferred aspect, this homologous recombination is carried out bytransforming embryo-derived stem (ES) cells with a vector containing theinsertionally inactivated MTSP protein gene, such that homologousrecombination occurs, followed by injecting the ES cells into ablastocyst, and implanting the blastocyst into a foster mother, followedby the birth of the chimeric animal (“knockout animal”) in which an MTSPprotein gene has been inactivated (see Capecchi, Science 244:1288-1292(1989)). The chimeric animal can be bred to produce additional knockoutanimals. Such animals can be mice, hamsters, sheep, pigs, cattle, etc.,and are preferably non-human mammals. In a specific embodiment, aknockout mouse is produced.

Such knockout animals are expected to develop or be predisposed todeveloping neoplastic diseases and thus can have use as animal models ofsuch diseases e.g., to screen for or test molecules for the ability totreat or prevent such diseases or disorders. Hence, the animal modelsfor are provided. Such an animal can be initially produced by promotinghomologous recombination between an MTSP gene in its chromosome and anexogenous MTSP protein gene that would be over-expressed ormis-expressed (preferably by expression under a strong promoter). In apreferred aspect, this homologous recombination is carried out bytransforming embryo-derived stem (ES) cells with a vector containing theover-expressed or mis-expressed MTSP protein gene, such that homologousrecombination occurs, followed by injecting the ES cells into ablastocyst, and implanting the blastocyst into a foster mother, followedby the birth of the chimeric animal in which an MTSP gene has beenover-expressed or mis-expressed (see Capecchi, Science 244:1288-1292(1989)). The chimeric animal can be bred to produce additional animalswith over-expressed or mis-expressed MTSP protein. Such animals can bemice, hamsters, sheep, pigs, cattle, etc., and are preferably non-humanmammals. In a specific embodiment, a mouse with over-expressed ormis-expressed MTSP protein is produced.

The following examples are included for illustrative purposes only andare not intended to limit the scope of the invention.

Example 1 Cloning of MTSP3, Cloning and Mutagenesis of the ProteaseDomain of MTSP3 1. Identification and Cloning of MTSP3

a. Identification of EST Clones AI924527 and AI924182 as Part of aSerine Protease MTSP3

DNA encoding the protease domain of the protease designated MTSP1 wasindependently cloned from the human prostatic adenocarcinoma cell line,PC-3, using degenerate oligonucleotide primers, then sequenced andcharacterized (see EXAMPLE 6). The sequence of the sense degenerateprimer used in cloning MTSP1 was 5′-TGGRT(I)VT(I)WS(I)GC(I)RC(I)CAYTG-3′(SEQ ID No: 13), and that of the anti-sense was5′-(I)GG(I)CC(I)CC(I)SWRTC(I)CCYT(I)RCA(I)GHRTC-3′ (SEQ ID No:14), whereR=A,G; V=G,A,C; W=A,T; S=G,C; Y=C,T; H=A,T,C. The primer sequencescorrespond to two highly conserved regions in all serine proteases andshould amplify PCR products ranging from 400 to 500 base pairs. MTSP1was subsequently found to be identical to matriptase (Genbank accessionnumber AF118224; see also Takeuchi et al., Proc. Natl. Acad. Sci. USA,96(20):11054-61 (1999); and Lin et al., J. Biol. Chem., 274(26):18231-61999).

Using the protein sequence of the protease domain of the serine proteaseMTSP1, the EST database (dbEST) at the National Center for BiotechnologyInformation (Bethesda, Md.; www.ncbi.nlm.nih.gov) was searched for ESTclones that contain similar or identical sequences to MTSP1 using thesearch algorithm tblastn. The tblastn algorithm compares a protein querysequence against a nucleotide sequence database dynamically translatedin all six reading frames (both strands). The sequences for twoidentical EST clones (NCI_CGAP_Lu19 AI924527 and AI924182) derived fromhuman lung tumor tissue showed 43% identity with the MTSP1 proteinsequence. Subsequent search of GenBank and SwissProt database for theEST sequence AI924527 and AI924182 did not show any matching sequence toMTSP1, indicating that the sequence contained in these EST clonesAI924527 and AI924182 may be portions of a new serine protease.

b. PCR Cloning of a cDNA Fragment of Another Membrane Type SerineProtease MTSP3

The double-stranded Marathon-Ready(tm) cDNA library derived from humanlung carcinoma (LX-1) was obtained from Clontech (Palo Alto, Calif.;catalog #7495-1) and used as a template. Two primers,5′-TCACCGAGAAGATGATGTGTGCAGGCATCC-3′ (SEQ ID No:15) (sense primer), and5′-GGGACAGGGGCTGTAAGGCAGGGAATGAG-3′ (SEQ ID No:16) (antisense primer),were used to amplify a ˜360 by DNA fragment. The PCR product wasseparated on a 2% agarose gel and purified using a gel extraction kit(catalog number 28706; QlAquick gel extraction kit; Qiagen). Thepurified DNA fragment was ligated into TA vectors (catalog numberK4500-01; TOPO-TA cloning kit, Invitrogen, Carlsbad, Calif.). Aftertransformation into E. coli cells, plasmids were isolated and analyzedby digestion with EcoRI restriction enzyme. Clones that had inserted DNAwere further characterized by sequencing using a fluorescent dye-basedDNA sequencing method (catalog number,4303149; BigDye terminator cyclesequencing kit with AmpliTaq DNA polymerase; Perkin Elmer, Lincoln,Calif.).

The DNA sequence obtained was analyzed and has 43% identity with theMTSP1 protein sequence. This indicates that the LX-1 cDNA librarycontains a desired nucleic acid molecule. It was used to isolate a cDNAclone encompassing a full length protease.

c. 5′- and 3′-Rapid Amplification of cDNA Ends (RACE)

To obtain the full-length cDNA that encoded this serine protease,hereafter called MTSP3, 5′- and 3′-RACE reactions were performed. TheMarathon-Ready cDNA library from human lung carcinoma (LX-1) was used toisolate the 5′ and 3′ ends of the cDNA encoding MTSP3. Marathon-ReadycDNA is specifically made for RACE reactions. Two gene specific primerswere used: 5′-CCCGCAGCCATAGCCCCAGCTAACG-3′ (SEQ ID No. 17) for 5′-RACEreaction and 5′-GCAGACGATGCGTACCAGGGGGAAGTC-3′ (SEQ ID No. 18) for3′-RACE reaction. Two fragments, approximately 1.8 kbp and 0.85 kbp,were isolated that correspond to the missing 5′ and 3′ end sequences,respectively. These fragments were subcloned as described above. Theywere further confirmed by Southern analysis using an internal cDNAfragment encompassing the 2 primers used in the RACE reactions as probeand by DNA sequence analysis.

d. PCR Amplification of cDNA Encoding Full-Length Protease Domain ofMTSP3

To obtain the cDNA fragment encoding the protease domain of MTSP3, anend-to-end PCR amplification using gene-specific primers was used. Thetwo primers used were: 5′-CTCGAGAAAAGAGTGGTGGGTGGGGAGGAGGCCTCTGTG-3′(SEQ ID No. 19) for the 5′ end and 5′-GCGGCCGCATTACAGCTCAGCCTTCCAGAC-3′(SEQ ID No. 20) for the 3′ end. The 5′ primer contains the sequence(underlined) that encodes the start of the MTSP3 protease domain(VVGGEEASV). The 3′ primer contains the stop codon (underlined) ofMTSP3. A ˜700-bp fragment was amplified and subcloned into a Pichiapastoris expression vector, pPIC9K.

e. C310S Mutagenesis of MTSP3

To eliminate the free cysteine (at position 310 in SEQ ID No. 4) thatexists when the protease domain of the MTSP3 protein is expressed or thezymogen is activated, the free cysteine at position 310 (see SEQ ID No.3), which is Cys122 if a chymotrypsin numbering scheme is used, wasreplaced with a serine. The resulting vector was designatedpPIC9K:MTSP3C122S.

The gene encoding the protease domain of MTSP3 was mutagenized by PCRSOE (PCR-based splicing by overlap extension) to replace the unpairedcysteine at position 310 (122 chymotrypsin numbering system) with aserine. Two overlapping gene fragments, each containing the TCT codonfor serine at position 310 were PCR amplified using the followingprimers: for the 5′ gene fragment,TCTCTCGAGAAAAGAGTGGTGGGTGGGTGGGGAGGAGGCCTCTGTG SEQ ID No. 51 andGCTCCTCATCAAAGAAGGGCAGAGAGATGGGCCTGACTGTGCC SEQ ID No. 52; for the 3′gene fragment, ATTCGCGGCCGCATTACAGCTCAGCCTTCCAGAC (SEQ ID No. 53) andGGCACAGTCAGGCCCATCTCTCTGCCCTTCTTTGATGAGGAGC (SEQ ID No. 54). Theamplified gene fragments were purified on a 1% agarose gel, mixed andreamplified by PCR to produce the full length coding sequence for MTSP3C122S. This sequence was then cut with restriction enzymes NotI andXhoI, and ligated into vector pPic9K.

2. Sequence Analysis

All derived DNA and protein sequences were analyzed using MacVector(version 6.5; Oxford Molecular Ltd., Madison, Wis.). The full-lengthcDNA encoding MTSP3 is composed of 2,137 base pairs containing thelongest open reading frame of 1,314 base pairs which translate to a437-amino acid protein sequence. The cDNA fragment (nt 873-1,574)encoding the protease domain of MTSP3 is composed of 702 base pairswhich translate to a 233-amino acid protein sequence plus the stopcodon. The DNA sequence and the translated protein sequence of MTSP3 areshown in SEQ ID Nos. 3 and 4, respectively.

3. Construction of the Expression Vectors

DNA encoding MTSP3 full length protein containing the C310S pointmutation (i.e., MTSP3C122S) was cloned from pPIC9K:MTSP3C122S. Theprimers MTSP3: 5′ GAATTCCATATGCCGCGCTTTAAAGTGGTGGGTGGGGAGGAGGCC SEQ IDNo. 47 (containing a NdeI restriction site) and MTSP3-3′GGGATACCCGTTACAGCTCAGCCTTCCAGAC 5′ SEQ ID No. 48 (containing a BamHIrestriction site) were used to PCR amplify the human MTSP3C122S proteasedomain utilizing a plasmin recognition sequence (PRFK) for zymogenactivation. Amplification was conducted in a total volume of 50 μlcontaining 10 mM KCl, 20 mM Tris-HCl (pH 8.8 at 25° C.), 10 mM(NH₄)₂SO₄, 2.0 mM MgSO₄, 0.1% Triton X-100, 0.3 mM dNTPs, 5.0 units ofvent DNA polymerase, and 100 pmol of primers. The reaction mixtures wereheated to 95° C. for 5 min, followed by 25-30 cycles of 95, 60, and 75°C. for 30 s each and a final extension at 75° C. for 2 min.

PCR products were purified using a QlAquick PCR purification kit (QIAGENInc., Chatsworth, Calif.). Full-length oligonucleotides were doublydigested with 10 units BamHI and 20 units NdeI for 2 h at 37° C. Thedigested fragments were purified on a 1.3% agarose gel and stained withethidium bromide. The band containing the MTSP3C122S encoding DNA wasexcised and purified using a QIAEX II gel extraction kit.

The MTSP3C122S encoding DNA was then cloned into the NdeI and BamHIsites of the pET19b vector (Novagen) using standard methods. This vectorallows the fusion of a HIS₆ tag for purification by metal affinitychromatography (MAC). Competent XL1 Blue cells (Stratagene) weretransformed with the pET19b-MTSP3C122S vector and used to produceplasmid stocks. Proper insertion and DNA sequence were confirmed byfluorescent thermal dye DNA sequencing methods as well as restrictiondigests.

4. Protein Expression, Purification, and Refolding

Overexpression of the gene product was achieved in E. coli strain BL21(DE3) (Novagen, Madison Wis.) containing the DNAY plasmid for rare codonoptimization (see, e.g., Garcia et al. (1986) Cell 45:453-459). Cellswere grown at 37° C. in (2×YT) media supplemented with carbenicillin andkanamycin to a final concentrations of 50 ug/ml and 34 ug/ml,respectively. One liter cultures were inoculated with 10 mL of anovernight culture grown in the same media. Cells were allowed to grow toa density of 0.6-1.0 OD₆₀₀ before the addition of IPTG (finalconcentration 1.0 mM). Cells were grown an additional 4 hours beforeharvesting.

The cell pellet was resuspended in 20 mL of lysis buffer (50 mM Na₂HPO₄,300 mM NaCl, pH 7.4). The cell suspension was treated with 10-20 mglysozyme and incubated at 37° C. for 1 hour. DNaseI was then added (1-2mg) with mixing until the solution was no longer viscous. The solutionwas then transferred to a Rosette flask and sonicated, on ice, at highpower for 15 min. Inclusion bodies were pelleted by centrifugation at20K rpm (˜48,000 g) at 4° C. for 30 min.

Inclusion bodies were washed by douncing 2 times in 50 mM Na₂HPO₄, 300mM NaCl, 5% LADO, pH 7.4 followed by 2 times in 50 mM Na₂HPO₄, 300 mMNaCl, pH 7.4. Inclusion bodies (˜500 mg) are solubilized in 25 mL 6 MGuHCl, 100 mM tris-HCl, 20 mM βMe, pH 8.0. This solution was spun at 20Krpm for 30 minutes to pull down any particulate matter. This solutionwas passed through a 0.2 μM filter and diluted to 100 mL insolubilization buffer.

MTSP3C122S was refolded by slowly adding the inclusion body mixture to 8L of refolding buffer (100 mM tris-HCl, 150 mM NaCl, 5 mM GSH, 0.05 mMGSSG, 1 M arginine, pH 8.0) using a peristaltic pump. The refoldingmixture was allowed to stir at 4° C. for 7 days or until the thiolconcentration was below 1 mM as detected by Ellman's reagent. Thesolution was filtered through a 5 μM filter, concentrated byultrafiltration and the buffer exchanged into MAC equilibration buffer(50 mM Na₂HPO₄, 300 mM NaCl, 10 mM imidazole, pH 8.0) by crossflowfiltration. The resulting solution was passed through a 0.2 μM filterand further purified on a FPLC (Amersham-Pharmacia) using Pharmaciachelating sepharose. The solution was loaded onto the nickel loaded MACat a flow rate of 1.0 mL/min and eluted with a linear gradient of 1.0mM-1.0 M imidazole in 50 mM Na₂HPO₄, 300 mM NaCl, pH 8.0. Proteincontaining fractions were determined by SDS-PAGE and subsequently pooledand frozen at −80° C.

Small amounts of purified MTSP3C122S were activated using plasminsepharose for 30 min. at 37° C. The resin was spun down at 14K rpm for 5min. and the protein solution removed. The resulting solution wasscreened for activity against of series of protease substrates;spec-tpa, spec-pl, spec-UK, spec-fXIIa (American Diagnostica), S-2238,S-2266 (Kabi Diagnostica), S-2586, S-2366, S-2444, S-2288, S-2251,S-2302, S-2765, S-2222, spec-THE (Chromogenix), spec-fVIIa (Pentapharm).MTSP3C122S cleaved several of these substrates efficiently but was mostactive towards Spec-fXIIa, Spec-tPA, S-2765, Spec-fVIIa and S-2444.

5. Gene Expression Profile of the Serine Protease MTSP3 in Normal andTumor Tissues

To obtain information regarding the tissue distribution of the MTSP3transcripts, the DNA insert encoding the MTSP3 protease domain was usedto probe a RNA blot composed of 76 different human tissues (catalognumber 7775-1; human multiple tissue expression (MTE) array; CLONTECH,Palo Alto, Calif.). The expression pattern observed in decreasing signallevel was: trachea=colon (descending)=esophagus>colon (ascending)>colon(transverse)=rectum>ileum>duodenum>jejunum>bladder>ilocecum>stomach>kidney>appendix.It is also expressed less abundantly in fetal kidney, and in two tumorcell lines, HeLa S3 and leukemia, K-562. Northern analysis using RNAblots (catalog numbers 7780-1, 7765-1 & 7782-1; human 12-lane, humanmuscle and human digestive system multiple tissue northern (MTN) blots;CLONTECH) confirmed that the expression was detected most abundantly inthe colon, moderately in the esophagus, small intestine, bladder andkidney, and less abundantly in stomach and rectum. A single transcriptof ˜2.2 kb was detected.

Amplification of the MTSP3 transcript in several human primary tumorsxenografted in mouse was performed using gene-specific primers. TheMTSP3 transcript was detected in lung carcinoma (LX-1), colonadenocarcinoma (CX-1), colon adenocarcinoma (GI-112) and ovariancarcinoma (GI-102). No apparent signal was detected in another form oflung carcinoma (GI-117), breast carcinoma (GI-101), pancreaticadenocarcinoma (GI-103) and prostatic adenocarcinoma (PC3).

Example 2 Identification of Genomic Clone of MTSP4

Using the nucleotide sequence encoding the protease domain of the serineprotease MTSP1 (also called matriptase), the protein database(SWISSPROT) at the National Center for Biotechnology Information(Bethesda, Md.; <http://www.ncbi.nlm.nih.gov>) was searched for similaror identical sequence to MTSP1 using the search algorithm blastx. Theblastx algorithm compares the six-frame conceptual translation productsof a nucleotide query sequence (both strands) against a protein sequencedatabase. A protein encoding sequence (CAA18442) that has 37% identityto the MTSP1 protein sequence that was found to include a putativeLDL-receptor domain and a trypsin-like serine protease domain wasidentified. This protein-encoding sequence (hereinafter referred to asMTSP4) was found to be encoded by a genomic clone (AL022314) derivedfrom human chromosome 22 sequenced by the Sanger Centre Chromosome 22Mapping Group and deposited into the public database as part of theHuman Genome Project. Subsequent search of the GenBank database showedthat no identical sequence has been deposited. A search of the ESTdatabase also did not show any matching human sequence, indicating thatno human EST clone exists in the public database. Mouse EST clones(AI391417 and AA208793) are present and showed 88% identity to theserine protease at the nucleotide level.

PCR Cloning of a Genomic DNA Fragment of MTSP4 for Use as HybridizationProbe

In order to obtain tissue distribution profile of MTSP4 as well as toidentify a tissue source for subsequent cloning of the cDNA, a genomicfragment was amplified from human genomic DNA using two gene-specificprimers, 5′-CCTCCACGGTGCTGTGGACCGTGTTCC-3′ (5′ primer) SEQ ID No. 21 and5′-CCTCGCGCAAGGCGCCCCAGCCCG-3′ (3′ primer) SEQ ID No. 22. These twoprimers amplified a 265-base pair fragment within a single exon ofMTSP4. The fragment was then used as a hybridization probe on humantissue northern blot (human 12-lane multiple tissue northern (MTN) blot(catalog number 7780-1); CLONTECH, Palo Alto, Calif.). A prominent band(˜2.6 kb) was detected in liver. Relatively weaker signals were obtainedfrom the brain, heart, skeletal muscle and kidney. Since human livershowed a very strong signal, this tissue was selected for theamplification of the MTSP4 cDNA.

5′- and 3′-Rapid Amplification of cDNA Ends (RACE)

To obtain a full-length clone encoding MTSP4, 5′- and 3′-RACE reactionswere performed. The Marathon-Ready cDNA library from human liver(CLONTECH) was used to isolate the 5′ and 3′ ends of the cDNA encodingMTSP4. Marathon-Ready cDNA clones are specifically made for RACEreactions. Two gene specific primers were used:5′-GCGTGGCGTCACCTGGTAGCGATAGACCTCGC-3′ (SEQ ID No. 23) for 5′-RACEreaction and 5′-CCTCCACGGTGCTGTGGACCGTGTTCC-3′ (SEQ ID No. 24) for3′-RACE reaction. No fragment was obtained from the initial 5′-RACEreaction.

The 3′-RACE reaction, however, produced a ˜1.5 kbp fragment. A nestedPCR reaction was used on the initial 5′-RACE reaction products to obtainpart of the 5′ end of MTSP4. The nested 5′ gene-specific primer used was5′-CCTCGCGCAAGGCGCCCCAGCCCG-3′ (SEQ ID No. 25) and produced a ˜0.8 kbpfragment. The fragments were subcloned into pCR2.1-TOPO TA cloningvector (Invitrogen, Carlsbad, Calif.). The resulting clones wereanalyzed by Southern analysis using the internal genomic fragmentencompassing the primers used in the RACE reactions as probe and by DNAsequence analysis. Sequence analysis of the 5′-RACE product showed thatthe potential initiation codon was still missing.

To obtain the 5′ cDNA end that encodes the N terminus of MTSP4, thepublicly available genomic sequence of chromosome 22 was searched forsequence corresponding to the sequence obtained in the 5′-RACE clone.The resulting genomic sequence was translated and the protein sequencewas compared to that derived from the translated sequence of the 5′-RACEclone. After determining the overlapping sequences, a gene-specificoligonucleotide primer (5′-TCATCGGCCAGAGGGTGATCAGTGAG-3′) SEQ ID No. 26corresponding to the sequence upstream of the potential initiation codonand another gene-specific oligonucleotide primer(5′-CCTCCTCAGTGCATAGGCATCAAACCAG-3′) SEQ ID No. 27 corresponding to asequence within the overlapping region were used to amplify the missing5′ cDNA of MTSP4 from the human liver cDNA library.

Splice Variants and Domain Organization of MTSP4

At least two cDNA fragments were consistently obtained during PCRamplification, indicating multiple splice variants of MTSP4. Subcloningand sequence analysis revealed that a longer, more abundant form,MTSP4-L and a shorter form, MTSP4-S. The encoded proteins aremulti-domain, type II membrane-type serine proteases and include atransmembrane domain at the N terminus followed by a CUB domain, 3 LDLRdomains and a trypsin-like serine protease domain at the C terminus. Thedifference between these two forms of MTSP4 is the absence in MTSP4-S ofa 432-bp nucleotide sequence between the transmembrane and the CUBdomains (see FIG. 2).

PCR Amplification of cDNA Encoding Full-Length Protease Domain of MTSP4

To obtain a cDNA fragment encoding the protease domain of MTSP4, anend-to-end PCR amplification using gene-specific primers and theMarathon-Ready cDNA library from human liver was used. The two primersused were: 5′-TCTCTCGAGAAAAGAATTGTTGGTGGAGCTGTGTCCTCCGAG-3′ (SEQ ID No.28) for the 5′ end and 5′-AGGTGGGCCTTGCTTTGCAGGGGGGCAGTTC-3′ for the 3′end SEQ ID NO. 29). The 5′ primer contained the sequence that encodesthe start of the MTSP4 protease domain (IVGGAVSSE). The 3′ primercorresponds to the sequence just downstream of the stop codon. A ˜740-bpfragment was amplified, subcloned into pCR2.1-TOPO TA cloning vector andsequenced.

Gene Expression Profile of MTSP4 in Normal and Tumor Tissues

To obtain information regarding the gene expression profile of the MTSP4transcript, a DNA fragment encoding part of the LDL receptor domain andthe protease domain was used to probe an RNA blot composed of 76different human tissues (catalog number 7775-1; human multiple tissueexpression (MTE) array; CLONTECH). As in the northern analysis of gelblot, a very strong signal was observed in the liver. Signals in othertissues were observed in (decreasing signal level): fetalliver>heart=kidney=adrenal gland=testis=fetal heart and kidney=skeletalmuscle=bladder=placenta>brain=spinal cord=colon=stomach=spleen=lymphnode=bone marrow=trachea=uterus=pancreas=salivary gland=mammarygland=lung. MTSP4 is also expressed less abundantly in several tumorcell lines including HeLa S3=leukemia K-562=Burkitt's lymphomas (Rajiand Daudi)=colorectal adenocarcinoma (SW480)>lung carcinoma(A549)=leukemia MOLT-4=leukemia HL-60. PCR of the MTSP4 transcript fromcDNA libraries made from several human primary tumors xenografted innude mice (human tumor multiple tissue cDNA panel, catalog numberK1522-1, CLONTECH) was performed using MTSP4-specific primers. The MTSP4transcript was detected in breast carcinoma (GI-101), lung carcinoma(LX-1), colon adenocarcinoma (GI-112) and pancreatic adenocarcinoma(GI-103). No apparent signal was detected in another form of lungcarcinoma (GI-117), colon adenocarcinoma (CX-1), ovarian carcinoma(GI-102). and prostatic adenocarcinoma (PC3). The MTSP4 transcript wasalso detected in LNCaP and PC-3 prostate cancer cell lines as well as inHT-1080 human fibrosarcoma cell line.

Sequence Analysis

MTSP4 DNA and protein sequences were analyzed using MacVector (version6.5; Oxford Molecular Ltd., Madison, Wis.). The ORF of MTSP4-L includes2,409 bp, which translate to a 802-amino acid protein, while the ORF ofMTSP4-S is composed of 1,977 by which translate to a 658-amino acidprotein. The cDNA encoding the protease domain in both forms is composedof 708 by which translate to a 235-amino acid protein sequence (see, SEQID No. 6) The DNA sequences and the translated protein sequences ofMTSP4-L and MTSP4-S, and of the protease domain of MTSP4 are set forthin SEQ ID Nos. 8, 10 and 6, respectively.

Example 3 Cloning of MTSP6

Identification of Genomic Clone of MTSP6

Using the protein sequence of the protease domain of the serine proteaseMTSP4 (see EXAMPLE 2), the non-redundant database (all non-redundantGenBank CDS translations+PDB+SwissProt+PIR+PRF) at the National Centerfor Biotechnology Information (Bethesda, Md.;<http://www.ncbi.nlm.nih.gov>) was searched for sequences that weresimilar or identical to MTSP4 using the search algorithm tblastn. Thetblastn algorithm compares a protein query sequence against a nucleotidesequence database dynamically translated in all reading frames. Aprotein (55 amino acids), which has 60% identity with the query MTSP4sequence (55 amino acids), was obtained from the translation of genomicsequence of AC015555 (nucleotide #15553 to 15717). This proteinhereafter is referred to as MTSP6. Subsequent search of the GenBankdatabase showed that no cDNA encoding MTSP6 has been deposited.

The gene exhibiting highest homology to MTSP6 was human transmembraneserine protease 2 (GenBank accession number U75329; Swissprot accessionnumber O15393), which showed 66% identity to MTSP6 within the 45 aminoacid regions compared. Consequently, the nucleotide sequence encodingthe MTSP6 protease domain was obtained by comparing the protein sequenceof human transmembrane serine protease 2 protease domain with thenucleotide sequence of AC015555 translated in six reading frames. Theprotein sequence obtained from the translated nucleotide sequence ofMTSP6 revealed an overall 50% identity with human transmembrane serineprotease 2. A search of the EST database indicated the presence of sevenMTSP6 EST clones (AA883068, AW591433, AI978874, AI469095, AI935487,AA534591 and AI758271).

Cloning of Human MTSP6 Full-Length cDNA

To obtain cDNA encoding the region of the MTSP6 protease domainidentified by database searches described above, two gene-specificprimers, Ch17-NSP-1, 5′-TCACGCATCGTGGGTGGAACATGTCC-3′ (5′ primer) SEQ IDNO. 30 nd Ch17-NSP-2AS, 5′-ACCCACCTCCATCTGCTCGTGGATCC-3′ SEQ ID NO. 31(3′ primer), were used for PCR. These two primers amplified a 708-basepair fragment from human mammary gland carcinoma cDNA (ClontechMarathon-Ready cDNA, Cat. No. 7493-1).

To obtain the remaining, unknown cDNA of MTSP6, 5′- and 3′-RACEreactions were performed on the human mammary gland carcinoma.Marathon-Ready cDNA is specifically made for RACE reactions. The firstRACE reactions were performed by PCR using Marathon cDNA adaptor primer1 (AP1) with gene specific primers, Ch17-NSP-2AS,5′-ACCCACCTCCATCTGCTCGTGGATCC-3′ SEQ ID NO. 31 for 5′-RACE reaction andCh17-NSP-1, 5′-TCACGCATCGTGGGTGGAACATGTCC-3′ SEQ ID NO. 30 for 3′-RACEreaction. The PCR products were purified from agarose gel. A secondnested PCR was then performed using Marathon cDNA adaptor primer 2 (AP2)with gene specific primer Ch17-NSP-3AS,5′-CCACAGCCTCCTCTCTTGACACACCAG-3′ SEQ ID No. 32 for 5′-RACE reaction(using first 5′-RACE product as template) and Ch17-NSP-35′-ACGCCCCTGTGGATCATCACTGCTGC-3′ SEQ ID No. 33 for 3′-RACE reaction(using first 3′-RACE product as template). First 5′- and 3′-RACEproducts were also used as template for PCR reactions using primersCh17-NSP-3 and Ch17-NSP-4AS to obtain a cDNA fragment for use as aprobe. PCR products from RACE reactions which were larger than 700 bywere cut out and purified from agarose gel and subcloned intopCR2.1-TOPO cloning vector (Invitrogen, Carlsbad, Calif.). Colonyhybridization was then performed to identify positive coloniescontaining MTSP6 sequence. Positive clones were identified by colonyhybridization using the 495 by DNA fragment obtained from PCR reaction(with primers Ch17-NSP-3 and Ch17-NSP-4AS) and by DNA sequencing.

Sequence analysis of the 5′-RACE products indicated that an additional420 by of upstream sequence were obtained. The potential initial codonwas not present in the 5′-RACE sequence. Another round of nested 5′-RACEreaction was performed using AP2 and a gene specific primer (designedbased on the new RACE sequence) Ch17-NSP-5AS5′-TCCCTCCCTCACATATACTGAGTGGTG-3′ SEQ ID No. 34, using the PCR productsobtained from the first 5′-RACE as template. A PCR product of 367 byusing Ch17-NSP-6 5′-CGACTGCTCAGGGAAGTCAGATGTCG-3′ SEQ ID NO. 35(designed based on the new 5′-RACE sequence) and Ch17-NSP-SAS was usedto identify the positive clones. An additional sequence of 480 by wasobtained from the second 5′-RACE products. A potential ATG start codonwas observed within a sequence of GTCACCATGG (nucleotides 262-272 of SEQID No. 12, which appears to be a Kozak sequence (GCC (A/G) CCAUGG),indicating that this ATG is likely the initiation codon for MTSP6.

The 3′-RACE reaction to obtain the rest of the 3′ end of MTSP6 was notsuccessful using Marathon Ready human mammary gland carcinoma cDNA. Thesequence of the 3′-RACE products obtained was exclusively that of anMTSP6 cDNA truncated with the Marathon AP2 primer sequence within thecoding region.

The 3′-end sequence of MTSP6 was obtained by PCR using Ch17-NSP-3(5′-ACGCCCCTGTGGATCATCACTGCTGC-3′; SEQ ID NO. 33) and Ch17-NSP-4(5′-CTGGTGTGTCAAGAGAGGAGGCTGTGG-3′; SEQ ID NO. 37) with an antisenseprimer Ch17-NSP-7AS (5′-ACTCAGGTGGCTACTTATCCCCTTCCTC-3; SEQ ID NO. 38)designed based on the sequence of an EST clone AA883068, whichapparently covers the 3′-end of MTSP6 sequence, and human smallintestine cDNA (Clontech) as template. Two PCR products (650 by and 182bp, respectively) were obtained and DNA sequence analysis indicated thatboth PCR products contained a stop codon.

Sequence Analysis and Domain Organization of MTSP6

The MTSP6 DNA and protein sequences were analyzed using DNA Strider(version 1.2). The ORF of MTSP6 is composed of 1,362 bp, which translateinto a 453-amino acid protein. Protein sequence analysis using the SMART(Simple Modular Architecture Research Tool) program athttp://smart.embl-heidelberg.de predicts that MTSP6 is a multi-domain,type-II membrane-type serine protease containing of a transmembranedomain (amino acids 48-68) at the N terminus followed by a LDLRa domain(LDL receptor domain class a) (amino acids 72-108), a SR domain(Scavenger receptor Cys-rich domain)(amino acids 109-205), and atrypsin-like serine protease domain (amino acids 216-443) (see FIG. 3).

Gene Expression Profile of MTSP6 in Normal and Tumor Tissues

To obtain information regarding the gene expression profile of the MTSP6transcript, a 495 by DNA fragment obtained from PCR reaction withprimers Ch17-NSP-3 and NSP-4AS was used to probe an RNA blot composed of76 different human tissues (catalog number 7775-1; human multiple tissueexpression (MTE) array; CLONTECH). The strongest signal was observed induodenum. Signal in other tissues were observed in (decreased signallevel): Stomach>trachea=mammary gland=thyroid gland=salivarygland=pituitarygland=pancreas>kidney>lung>jejunum=ileum=ilocecum=appendix=fetalkidney>fetal lung. Very weak signals can also be detected in severalother tissues. MTSP6 is also expressed in several tumor cell linesincluding HeLa S3>colorectal adenocarcinoma (SW480)>leukemiaMOLT-4>leukemia K-562. PCR analysis of the MTSP6 transcript from cDNAlibraries made from several human primary tumors xenografted in nudemice (human tumor multiple tissue cDNA panel, catalog number K1522-1,CLONTECH) was performed using MTSP6-specific primers (Ch17-NSP-3 andCh17-NSP2AS). The MTSP6 transcript was strongly detected in lungcarcinoma (LX-1), moderately detected in pancreatic adenocarcinoma(GI-103), weakly detected in ovarian carcinoma (GI-102); and very weaklydetected in colon adenocarcinoma (GI-112 and CX-1), breast carcinoma(GI-101), lung carcinoma (GI-117) and prostatic adenocarcinoma (PC3).The MTSP6 transcript was also detected in breast cancer cell lineMDA-MB-231, prostate cancer cell line PC-3, but not in HT-1080 humanfibrosarcoma cell line. MTSP6 is also expressed in mammary glandcarcinoma cDNA (Clontech).

Example 4 Expression of the Protease MTSP Domains

The DNA encoding each of the MTSP 3 and 4 protease domains was clonedinto a derivative of the Pichia pastoris vector pPIC9K (available fromInvitrogen; see SEQ ID NO. 45). Plasmid pPIC9k features include the 5′AOX1 promoter fragment at 1-948; 5′ AOX1 primer site at 855-875;alpha-factor secretion signal(s) at 949-1218; alpha-factor primer siteat 1152-1172; multiple cloning site at 1192-1241; 3′ AOX1 primer site at1327-1347; 3′ AOX1 transcription termination region at 1253-1586; HIS4ORF at 4514-1980; kanamycin resistance gene at 5743-4928; 3′ AOX1fragment at 6122-6879; ColE1 origin at 7961-7288; and the ampicillinresistance gene at 8966-8106. The plasmid used herein is derived frompPIC9K by eliminating the XhoI site in the kanamycin resistance gene andthe resulting vector is herein designated pPIC9KX.

Primers Used for PCR Amplification of Protease Domain and Subcloninginto the XhoI/NotI Sites of Pichia Vector MTSP3

5′ primer (with XhoI site [underlined]) SEQ ID No. 395′ TCTCTCGAGAAAAGAGTGGTGGGTGGGGAGGAGGCCTCTGTG 3′ 3′primer (with NotIsite [underlined]) SEQ ID No. 40 5′ ATTCGCGGCCGCATTACAGCTCAGCCTTCCAGAC3′

MTSP4-S and MTSP4-L

5′ primer (with XhoI site [underlined]) SEQ ID No. 415′ TCTCTCGAGAAAAGAATTGTTGGTGGAGCTGTGTCCTCCGAG 3′ primer with NotI siteSEQ ID No. 42 5′ ATTCGCGGCCGCTCAGGTCACCACTTGCTGGATCCAG 3′

MTSP6

MTSP6 was cloned into the E. coli TOPO vector (pcR® 2.1 TOPO™, SEQ IDNo. 46, Invitrogen, Carlsbad, Calif.; the TOPO® TA Cloning® Kit isdesigned form cloning Taq-amplified PRCR products).

5′ primer (with XhoI site [underlined]) SEQ ID No. 435′ CTCGAGAAACGCATCGTGGGTGGAAACATGTCCTTG 3′ 3′ primer NotI site comesfrom E. coli TOPO vector SEQ ID No. 44: 5′ ACTCAGGTGGCTACTTATCCCCTTCCTC3′

Example 5

Assays for Identification of Candidate Compounds that Modulate thatActivity of an MTSP

Assay for Identifying Inhibitors

The ability of test compounds to act as inhibitors of catalytic activityof an MTSP, including MTSP1, MTSP3, MTSP4, MTSP6 can be assessed in anamidolytic assay. The inhibitor-induced inhibition of amidolyticactivity by a recombinant MTSP or the protease domain portions thereof,can be measured by IC50 values in such an assay.

An exemplary assay buffer is HBSA (10 mM Hepes, 150 mM sodium chloride,pH 7.4, 0.1% bovine serum albumin). All reagents were from SigmaChemical Co. (St. Louis, Mo.), unless otherwise indicated. Two IC50assays at 30-minute (a 30-minute preincubation of test compound andenzyme) and at 0-minutes (no preincubation of test compound and enzyme)are conducted. For the IC50 assay at 30-minute, the following reagentsare combined in appropriate wells of a Corning microtiter plate: 50microliters of HBSA, 50 microliters of the test compound, diluted(covering a broad concentration range) in HBSA (or HBSA alone foruninhibited velocity measurement), and 50 microliters of the MTSP orprotease domain thereof diluted in buffer, yielding a final enzymeconcentration of about 100-500 pM. Following a 30-minute incubation atambient temperature, the assay is initiated by the addition of 50microliters of a substrate for the particular MTSP (see, e.g., table anddiscussion below) and reconstituted in deionized water, followed bydilution in HBSA prior to the assay) were added to the wells, yielding afinal volume of 200 microliters and a final substrate concentration of300 μM (about 1.5-times Kin).

For an 1050 assay at 0-minute, the same reagents are combined: 50microliters of HBSA, 50 microliters of the test compound, diluted(covering the identical concentration range) in HBSA (or HBSA alone foruninhibited velocity measurement), and 50 microliters of the substrate,such as a chromogenic substrate. The assay is initiated by the additionof 50 microliters of MTSP. The final concentrations of all componentsare identical in both 1050 assays (at 30- and 0-minute incubations).

The initial velocity of the substrate hydrolysis is measured in bothassays by, for example for a chromogenic substrate, as the change ofabsorbance at a particular wavelength, using a Thermo Max KineticMicroplate Reader (Molecular Devices) over a 5 minute period, in whichless than 5% of the added substrate was used. The concentration of addedinhibitor, which caused a 50% decrease in the initial rate of hydrolysiswas defined as the respective 1050 value in each of the two assays (30-and 0-minute).

Another Assay for Identifying Inhibitors

Test compounds for inhibition of the protease activity of the proteasedomain of is assayed in Costar 96 well tissue culture plates (CorningN.Y.). Approximately 2-3 nM the MTSP or protease domain thereof is mixedwith varying concentrations of inhibitor in 29.2 mM Tris, pH 8.4, 29.2mM imidazole, 217 mM NaCl (100 mL final volume), and allowed to incubateat room temperature for 30 minutes. 400 mM substrate is added, and thereaction monitored in a SpectraMAX Plus microplate reader (MolecularDevices, Sunnyvale Calif.) by following the change in a parametercorrelated with hydrolysis, such as absorbance for a chromogenicsubstrate for 1 hour at 37° C.

Assay for Screening MTSP6

The protease domain of MTSP6 expressed in Pichia pastoris is assayed forinhibition by various compounds in Costar 96 well tissue culture plates(Corning N.Y.). Approximately 1-20 nM MTSP6 is mixed with varyingconcentrations of inhibitor in 29.2 mM Tris, pH 8.4, 29.2 mM Imidazole,217 mM NaCl (100 μL final volume), and allowed to incubate at roomtemperature for 30 minutes. 500 μM substrate Spectrozyme t-PA (AmericanDiagnostica, Greenwich, Conn.) is added, and the reaction is monitoredin a SpectraMAX Plus microplate reader (Molecular Devices, SunnyvaleCalif.) by measuring the change in absorbance at 405 nm for 30 minutesat 37° C.

Identification of Substrates

Particular substrates for use in the assays can be identifiedempirically by testing substrates. The following list of substrates areexemplary of those that can be tested.

Substrate name Structure S 2366 pyroGlu-Pro-Arg-pNA•HCl spectrozyme t-PACH₃SO₂-D-HHT-Gly-Arg-pNA•AcOH N-p-tosyl-Gly-Pro-N-p-tosyl-Gly-Pro-Arg-pNA Arg-pNA Benzoyl-Val-Gly-Benzoyl-Val-Gly-Arg-pNA Arg-pNA Pefachrome t-PA CH₃SO₂-D-HHT-Gly-Arg-pNAS 2765 N-α-Z-D-Arg-Gly-Arg-pNA•2HCl S 2444 pyroGlu-Gly-Arg-pNA•HCl S2288 H-D-Ile-Pro-Arg-pNA•2HCl spectrozyme UKCbo-L-(γ)Glu(α-t-BuO)-Gly-Arg-pNA•2AcOH S 2302 H-D-Pro-Phe-Arg-pNA•2HClS 2266 H-D-Val-Leu-Arg-pNA•2HCl S 2222 Bz-Ile-Glu(g-OR)-Gly-Arg-pNA•HClR = H(50%) and R = CH₃(50%) Chromozyme PK Benzoyl-Pro-Phe-Arg-pNA S 2238H-D-Phe-Pip-Arg-pNA•2HCl S 2251 H-D-Val-Leu-Lys-pNA•2HCl Spectrozyme PlH-D-Nle-HHT-Lys-pNA•2AcOH Pyr-Arg-Thr-Lys-Arg-AMC H-Arg-Gln-Arg-Arg-AMCBoc-Gln-Gly-Arg-AMC Z-Arg-Arg-AMC Spectrozyme THEH-D-HHT-Ala-Arg-pNA•2AcOH Spectrozyme fXIIa H-D-CHT-Gly-Arg-pNA•2AcOHCVS 2081-6 (MeSO₂-dPhe-Pro-Arg-pNA) Pefachrome fVIIa(CH₃SO₂-D-CHA-But-Arg-pNA) pNA = para-nitranilide (chromogenic) AMC =amino methyl coumarin (fluorescent)

If none of the above substrates are cleaved, a coupled assay, describedabove, can be used. Briefly, test the ability of the protease toactivate and enzyme, such as plasminogen and trypsinogen. To performthese assays, the single chain protease is incubated with a zymogen,such as plasminogen or trypsinogen, in the presence of the a knownsubstrate, such, lys-plasminogen, for the zymogen. If the single chainactivates the zymogen, the activated enzyme, such as plasmin andtrypsin, will degrade the substrate therefor.

Example 6 Isolation and Cloning of Matriptase

A. Cell Type and Growth of Cells

Human prostate adenocarcinoma cell line, PC-3, was purchased from ATCC(catalog number CRL-1435; Manassas, Va.). The cells were cultured at 37°C., 5% CO₂ in Ham's F-12K growth medium (catalog number 9077; Irvine)supplemented with 2 mM L-glutamine and 10% fetal bovine serum. Allsubsequent cell manipulations were carried out according to themanufacturer's instructions. PC-3 cells were allowed to grow to about90% confluence, and were then washed briefly with 1× phosphate bufferedsaline.

B. Isolation of Total RNA, and Purification and Enrichment of polyA+ RNA

PC-3 cells were lysed in Trizol reagent (catalog number 15596; LifeTechnologies, Rockville, Md.) and total RNA was isolated according tothe manufacturer's protocol. The concentration of total RNA wasestimated from absorbance reading at 260 nm. PolyA⁺ RNA was purified andenriched using oligo-dT beads (catalog number 70061; Oligotex, Qiagen,Valencia, Calif.).

C. Reverse-Transcription and Polymerase Chain Reaction (PCR)

PC-3-derived polyA⁺ RNA was converted to single-stranded cDNA (sscDNA)by reverse transcription using ProSTAR first-strand RT-PCR kit (catalognumber 200420; Stratagene, La Jolla, Calif.) and SuperScript II RNase H-reverse transcriptase (catalog number 18064-022; Life Technologies).After reverse transcription, an aliquot of PC-3 sscDNA (4 μL) wassubjected to PCR using 2 mM each of the sense and anti-sense degenerateoligonucleotide primers and Taq polymerase (catalog number 201203;Qiagen). Total reaction volume was 100 μL. The sequence of the senseprimer was 5′TGGRT(I)VT(I)WS(I)GC(I)RC(I)CAYTG-3′ (SEQ ID No. 13) andthat of the anti-sense was5′(I)GG(I)CC(I)CC(I)SWRTC(I)CCYT(I)RCA(I)GHRTC-3′ (SEQ ID No. 14), whereR=A,G; V=G,A,C; W=A,T; S=G,C; Y=C,T; H=A,T,C. The primer sequencescorrespond to two highly conserved regions in all chymotrypsin-likeserine proteases and amplify PCR products ranging from approximately 400to 500 base pairs.

D. Clone Screening and Sequencing

The PCR products were separated on a 2% agarose gel and purified using agel extraction kit (catalog number 28706; QIAquick gel extraction kit;Qiagen). The purified DNA fragments were ligated into pCR2.1-TOPO(catalog number K4500-01; Invitrogen, Carlsbad, Calif.). Aftertransformation into E. coli cells, plasmid DNA was isolated and analyzedby digestion with EcoRI restriction enzyme. Clones that had insertednucleic acid were further characterized by sequencing using afluorescent dye-based DNA sequencing method (catalog number 4303149;BigDye terminator cycle sequencing kit with AmpliTaq DNA polymerase;Perkin Elmer, Lincoln, Calif.). A total of 31 clones were sequenced andanalyzed. All sequences were analyzed by a multiple nucleotide sequencealignment algorithm (blastn) (www.ncbi.nlm.nih.gov/blast) to identifyidentical or closely related DNA deposited in GenBank (NCBI, Bethesda,Md.). Those that did not show significant homology were further analyzedusing blastx, which compares the six-frame conceptual translationproducts of a nucleotide sequence (both strands) against a proteinsequence database (SwissProt). Eight clones yielded identical cDNAfragments that encode MTSP1. MTSP1 was subsequently found to beidentical to matriptase (GenBank accession number AF 118224).

E. Rapid Amplification of cDNA Ends (RACE) and Gene-SpecificAmplification of MTSP1

To obtain DNA encoding the complete protease domain of MTSP1, RACE andgene-specific amplification reactions were performed. A human prostateMarathon-Ready cDNA (catalog #7418-1; Clontech) was used to isolate partof the cDNA encoding MTSP1. Marathon-Ready cDNA is prepared to contain aknown hybridization sequence at the 5′ and 3′ ends of the sscDNA. The 3′region of MTSP1 cDNA was obtained by a 3′-RACE reaction using a genespecific primer, 5′-CACCCCTTCTTCAATGACTTCACCTTCG-3′ (SEQ ID No. 55). The5′ end of the MTSP 1 protease domain was obtained by gene-specificamplification reaction using two MTSP1-specific primers,5′-TACCTCTCCTACGACTCC-3′ (SEQ ID No. 56) for the sense primer and5′-GAGGTTCTCGCAGGTGGTCTGGTTG-3′ (SEQ ID No. 57) for the antisenseprimer. The sequences for these two primers were obtained from the humanSNC19 mRNA sequence. The 3′-RACE reaction and gene-specific PCR producedDNA fragments that were >1 kbp in size. These fragments were subclonedinto pCR2.1-TOPO (Invitrogen, San Diego, Calif.). After transformationinto E. coli cells, plasmid DNA was isolated and analyzed by digestionwith EcoRI restriction enzyme. Clones that had inserts werecharacterized by Southern blot analysis (using the internal cDNAfragment as probe) and by DNA sequence analysis.

F. PCR Amplification of cDNA Encoding the Protease Domain of MTSP1

To obtain a cDNA fragment encoding the entire protease domain of MTSP 1,an end-to-end PCR amplification using gene-specific primers was used.The two primers used were: 5′-CTCGAGAAAAGAGTTGTTGGGGGCACGGATGCGGATGAG-3′(SEQ ID No. 58) for the 5′ end and5′-GCGGCCGCACTATACCCCAGTGTTCTCTTTGATCCA-3′ (SEQ ID No. 36 for the 3′end. The 5′ primer contained the sequence that encodes the start of theMTSP1 protease domain (VVGGTDADE) (SEQ. ID. NO. 10). The 3′ primercontained the stop codon of MTSP 1. A ˜800-bp fragment was amplified,purified and subcloned into the Pichia pastoris expression vector,pPIC9K, resulting in pPIC9K-MTSP 1.

G. Gene Expression Profile of MTSP1 in Normal Tissues, Cancer Cells andCancer Tissues

To obtain information regarding the tissue distribution and geneexpression level of MTSP1, the DNA insert from pPIC9K-MTSP1 was used toprobe a blot containing RNA from 76 different human tissues (catalognumber 7775-1; human multiple tissue expression (MTE) array; CLONTECH,Palo Alto, Calif.). Significant expression was observed in the colon(ascending, transverse and descending), rectum, trachea, esophagus andduodenum. Moderate expression levels were observed in the jejunum,ileum, ilocecum, stomach, prostate, pituitary gland, appendix, kidney,lung, placenta, pancreas, thyroid gland, salivary gland, mammary gland,fetal kidney, and fetal lung. Lower expression levels were seen in thespleen, thymus, peripheral blood leukocyte, lymph node, bone marrow,bladder, uterus, liver, adrenal gland, fetal heart, fetal liver, fetalspleen, and fetal thymus. A significant amount of the MTSP1 transcriptwas also detected in colorectal adenocarcinoma cell line (SW480),Burkitt's lymphoma cell line (Daudi), and leukemia cell line (HL-60).RT-PCR of the MTSP1 transcript in several human primary tumorsxenografted in athymic nude mice was performed using gene-specificprimers. A high level of MTSP1 transcript was detected in colonadenocarcinoma (CX-1) and pancreatic adenocarcinoma (GI-103). Moderatelevels were observed in another colon adenocarcinoma (GI-112), ovariancarcinoma (GI-102), lung carcinoma (LX-1), and breast carcinoma(GI-101). Another lung carcinoma (GI-117) expressed a low level of theMTSP1 transcript. A similar RT-PCR was performed to detect the presenceof the MTSP1 transcript in PC-3 and LNCaP cell lines. Both cell linesexpressed significant amounts of MTSP1 transcript.

H. Sequence Analysis

All derived DNA and protein sequences were analyzed using MacVector(version 6.5; Oxford Molecular Ltd., Madison, Wis.). The cDNA encodingthe protease domain of MTSP1 is composed of 726 base pairs whichtranslate into a 241-amino acid protein sequence (rMAP) (see SEQ ID No.1, 2, 49 and 50).

Example 7

Production of Recombinant Serine Protease Domain of Matriptase or MTSP1(rMAP)

A. Fermentation

The production of multi-milligram amounts of rMAP was carried out byfermentation in a BioFlo 3000 fermentor (New Brunswick Scientific, N.J.)equipped with a 3.3 L capacity bioreactor using a SMD1168/pPIC9K:MTSP1Sac SC1 clone. ZA001 complex media (10 g/L yeast extract, 20 g/Lpeptone, 40 g/L glycerol, 5 g/L ammonium sulfate, 0.2 g/L calciumsulfate dihydrate, 2 g/L magnesium sulfate heptahydrate, 2 g/L potassiumsulfate, 25 g/L sodium hexametaposphate, 4.35 ml/L PTM1) was inoculatedwith 100 ml of an overnight culture of the P. pastoris transformant. Theculture was supplemented with 50% glycerol by fed-batch phase andinduced for 18-24 hours with methanol controlled at 0.025%.

B. Purification of Recombinant Serine Protease Domain of Matriptase orMTSP1 (rMAP)

The rMAP was secreted into the culture medium, so the first step of thepurification involved the removal of cells and cell debris bycentrifugation at 5000 g for 30 minutes. The resulting supernatant wasdecanted, adjusted to pH 8.0 with 10 N NaOH, and filtered through aSartoBran 300 0.45+0.2 μM capsule. This supernatant was concentrated to1 L by ultrafiltration using a 10 kDa ultrafiltration cartridge (NC SRTUF system with AG/Technologies UFP-10-C-5A filter), and the buffer wasexchanged by crossflow filtration into 50 mM tris-HCl, 50 mM NaCl, 0.05%tween-80, pH 8.0 (buffer A). The filtration unit was rinsed once with 1L buffer A which was combined with the concentrate.

The concentrated rMAP-containing solution was passed over a 150 mlbenzamidine column that had been equilibrated with buffer A, at a flowrate of 8 ml/min. The column was washed with 3 column volumes of 50 mMtris-HCl, 1.0 M NaCl, 0.05% tween-80, pH 8.0 (buffer B) and eluted with3 column volumes of 50 mM tris-HCl, 1.0 M L-arginine, 0.05% tween-80, pH8.0 (buffer C). Fractions containing rMAP were identified by activityassay and pooled. This pooled material was concentrated to 10 ml using aJumboSep concentrator (Pall Gelman) and a 10 kDa cutoff membrane. Onceconcentrated to 10 ml, the buffer was exchanged into 50 mM Na₂HPO₄, 125mM NaCl, pH 5.5 (buffer D) and the volume adjusted to 5-10 ml. Theretentate was removed and the concentrator washed with buffer D whichwas added to the concentrate. The total sample volume was adjusted 15ml.

The partially purified rMAP was passed through a 5 ml Q-sepharose FastFlow HiTrap column (Amersham-Pharmacia Biotech) pre-equilibrated with 15ml of buffer D. The flow through was collected. The HiTrap column waswashed with an additional 10 ml of buffer D. Both flow throughs werepooled, and the protein concentration was determined by measurement ofOD₂₈₀ (using an extinction coefficient of 2.012 mg/OD₂₈₀). Purified rMAPwas then deglycosylated by the addition 0.1 μl of Endoglycosidase H(ProZyme, 5 U/ml) per mg of protein and incubating overnight at 4° C.with gentle swirling.

The conductivity of the deglycosylated pool was adjusted to 2.0-3.0mS/cm with Nanopure H₂O and the pH adjusted to 6.5 (−200-300 mL finalvolume). The rMAP was then further purified by anion exchangechromatography by loading directly onto a Pharmacia Akta Explorer systemusing a 7 mL Source 15Q anion exchange column (Amersham-PharmaciaBiotech). The protein was eluted in a buffer containing 50 mM HEPES, pH6.5 with a 0-0.33 M NaCl gradient over 10 column volumes at a flow rateof 6 ml/min. Fractions containing protein were pooled, and benzamidinewas added to a final concentration of 10 mM. Protein purity was examinedby SDS-PAGE and protein concentration determined by measurement of OD₂₈₀and use of a theoretical extinction coefficient of 2.012 mg/OD₂₈₀.

Example 8 Assays

Amidolytic Assay for Determining Inhibition of Serine Protease Activityof Matriptase or MTSP1

The ability of test compounds to act as inhibitors of rMAP catalyticactivity was assessed by determining the inhibitor-induced inhibition ofamidolytic activity by the MAP, as measured by IC₅₀ values. The assaybuffer was HBSA (10 mM Hepes, 150 mM sodium chloride, pH 7.4, 0.1%bovine serum albumin). All reagents were from Sigma Chemical Co. (St.Louis, Mo.), unless otherwise indicated.

Two IC₅₀ assays (a) one at either 30-minutes or 60-minutes (a 30-minuteor a 60-minute preincubation of test compound and enzyme) and (b) one at0-minutes (no preincubation of test compound and enzyme) were conducted.For the IC₅₀ assay at either 30-minutes or 60-minutes, the followingreagents were combined in appropriate wells of a Corning microtiterplate: 50 microliters of HBSA, 50 microliters of the test compound,diluted (covering a broad concentration range) in HBSA (or HBSA alonefor uninhibited velocity measurement), and 50 microliters of the rMAP(Corvas International) diluted in buffer, yielding a final enzymeconcentration of 250 pM as determined by active site filtration.Following either a 30-minute or a 60-minute incubation at ambienttemperature, the assay was initiated by the addition of 50 microlitersof the substrate S-2765(N-α-Benzyloxycarbonyl-D-arginyl-L-glycyl-L-arginine-p-nitroanilinedihydrochloride; DiaPharma Group, Inc.; Franklin, Ohio) to each well,yielding a final assay volume of 200 microliters and a final substrateconcentration of 100 μM (about 4-times K_(m)). Before addition to theassay mixture, S-2765 was reconstituted in deionized water and dilutedin HBSA. For the IC₅₀ assay at 0 minutes; the same reagents werecombined: 50 microliters of HBSA, 50 microliters of the test compound,diluted (covering the identical concentration range) in HBSA (or HBSAalone for uninhibited velocity measurement), and 50 microliters of thesubstrate S-2765. The assay was initiated by the addition of 50microliters of rMAP. The final concentrations of all components wereidentical in both IC₅₀ assays (at 30- or 60- and 0-minute).

The initial velocity of chromogenic substrate hydrolysis was measured inboth assays by the change of absorbance at 405 nM using a Thermo Max®Kinetic Microplate Reader (Molecular Devices) over a 5 minute period, inwhich less than 5% of the added substrate was used. The concentration ofadded inhibitor, which caused a 50% decrease in the initial rate ofhydrolysis was defined as the respective IC₅₀ value in each of the twoassays (30- or 60-minutes and 0-minute).

In vitro Enzyme Assays for Specificity Determination

The ability of compounds to act as a selective inhibitor of matriptaseactivity was assessed by determining the concentration of test compoundthat inhibits the activity of matriptase by 50%, (IC₅₀) as described inthe above Example, and comparing IC₅₀ value for matriptase to thatdetermined for all or some of the following serine proteases: thrombin,recombinant tissue plasminogen activator (rt-PA), plasmin, activatedprotein C, chymotrypsin, factor Xa and trypsin.

The buffer used for all assays was HBSA (10 mM HEPES, pH 7.5, 150 mMsodium chloride, 0.1% bovine serum albumin). The assay for IC₅₀determinations was conducted by combining in appropriate wells of aCorning microtiter plate, 50 microliters of HBSA, 50 microliters of thetest compound at a specified concentration (covering a broadconcentration range) diluted in HBSA (or HBSA alone for V₀ (uninhibitedvelocity) measurement), and 50 microliters of the enzyme diluted inHBSA. Following a 30 minute incubation at ambient temperature, 50microliters of the substrate at the concentrations specified below wereadded to the wells, yielding a final total volume of 200 microliters.The initial velocity of chromogenic substrate hydrolysis was measured bythe change in absorbance at 405 nm using a Thermo Max® KineticMicroplate Reader over a 5 minute period in which less than 5% of theadded substrate was used. The concentration of added inhibitor whichcaused a 50% decrease in the initial rate of hydrolysis was defined asthe IC₅₀ value.

Thrombin (fIIa) Assay

Enzyme activity was determined using the chromogenic substrate,Pefachrome t-PA(CH₃SO₂-D-hexahydrotyrosine-glycyl-L-Arginine-p-nitroaniline, obtainedfrom Pentapharm Ltd.). The substrate was reconstituted in deionizedwater prior to use. Purified human aqhrombin was obtained from EnzymeResearch Laboratories, Inc. The buffer used for all assays was HBSA (10mM HEPES, pH 7.5, 150 mM sodium chloride, 0.1% bovine serum albumin).

IC₅₀ determinations were conducted where HBSA (50 μL), α-thrombin (50μl) (the final enzyme concentration is 0.5 nM) and inhibitor (50 μl)(covering a broad concentration range), were combined in appropriatewells and incubated for 30 minutes at room temperature prior to theaddition of substrate Pefachrome-t-PA (50 μl) (the final substrateconcentration is 250 μM, about 5 times Km). The initial velocity ofPefachrome t-PA hydrolysis was measured by the change in absorbance at405 nm using a Thermo Max® Kinetic Microplate Reader over a 5 minuteperiod in which less than 5% of the added substrate was used. Theconcentration of added inhibitor which caused a 50% decrease in theinitial rate of hydrolysis was defined as the IC₅₀ value.

Factor Xa

Factor Xa catalytic activity was determined using the chromogenicsubstrate S-2765(N-benzyloxycarbonyl-D-arginine-L-glycine-L-arginine-p-nitroaniline),obtained from DiaPharma Group (Franklin, Ohio). All substrates werereconstituted in deionized water prior to use. The final concentrationof S-2765 was 250 μM (about 5-times Km). Purified human Factor X wasobtained from Enzyme Research Laboratories, Inc. (South Bend, Ind.) andFactor Xa (FXa) was activated and prepared from it as described [Bock,P. E., Craig, P. A., Olson, S. T., and Singh, P. Arch. Biochem. Biophys.273:375-388 (1989)]. The enzyme was diluted into HBSA prior to assay inwhich the final concentration was 0.25 nM.

Recombinant Tissue Plasminogen Activator (rt-PA) Assay

rt-PA catalytic activity was determined using the substrate, Pefachromet-PA (CH₃SO₂-D-hexahydrotyrosine-glycyl-L-arginine-p-nitroaniline,obtained from Pentapharm Ltd.). The substrate was made up in deionizedwater followed by dilution in HBSA prior to the assay in which the finalconcentration was 500 micromolar (about 3-times Km). Human rt-PA(Activase®) was obtained from Genentech Inc. The enzyme wasreconstituted in deionized water and diluted into HBSA prior to theassay in which the final concentration was 1.0 nM.

Plasmin Assay

Plasmin catalytic activity was determined using the chromogenicsubstrate, S-2366 [L-pyroglutamyl-L-prolyl-L-arginine-p-nitroanilinehydrochloride], which was obtained from DiaPharma group. The substratewas made up in deionized water followed by dilution in HBSA prior to theassay in which the final concentration was 300 micromolar (about2.5-times Km). Purified human plasmin was obtained from Enzyme ResearchLaboratories, Inc. The enzyme was diluted into HBSA prior to assay inwhich the final concentration was 1.0 nM.

Activated Protein C (aPC) Assay

aPC catalytic activity was determined using the chromogenic substrate,Pefachrome PC(delta-carbobenzloxy-D-lysine-L-prolyl-L-arginine-p-nitroanilinedihydrochloride), obtained from Pentapharm Ltd.). The substrate was madeup in deionized water followed by dilution in HBSA prior to the assay inwhich the final concentration was 400 micromolar (about 3-times Km).Purified human aPC was obtained from Hematologic Technologies, Inc. Theenzyme was diluted into HBSA prior to assay in which the finalconcentration was 1.0 nM.

Chymotrypsin Assay

Chymotrypsin catalytic activity was determined using the chromogenicsubstrate, S-2586(methoxy-succinyl-L-arginine-L-prolyl-L-tyrosyl-p-nitroanilide), whichwas obtained from DiaPharma Group. The substrate was made up indeionized water followed by dilution in HBSA prior to the assay in whichthe final concentration was 100 micromolar (about 9-times Km). Purified(3X-crystallized; CDI) bovine pancreatic alpha-chymotrypsin was obtainedfrom Worthington Biochemical Corp. The enzyme was reconstituted indeionized water and diluted into HBSA prior to assay in which the finalconcentration was 0.5 nM.

Trypsin Assay

Trypsin catalytic activity was determined using the chromogenicsubstrate, S-2222 (benzoyl-L-isoleucine-L-glutamic acid-[gamma-methylester]-L-arginine-p-nitroanilide), which was obtained from DiaPharmaGroup. The substrate was made up in deionized water followed by dilutionin HBSA prior to the assay in which the final concentration was 250micromolar (about 4-times Km). Purified (3×-crystallized; TRL3) bovinepancreatic trypsin was obtained from Worthington Biochemical Corp. Theenzyme was reconstituted in deionized water and diluted into HBSA priorto assay in which the final concentration was 0.5 nM.

Since modifications will be apparent to those of skill in this art, itis intended that this invention be limited only by the scope of theappended claims.

1. A nucleic acid molecule, comprising a sequence of nucleic acidsencoding a single chain polypeptide that consists only of a proteasedomain of a type-II membrane-type serine protease (MTSP), or aproteolytically active fragment thereof, as a single chain, wherein: afree Cys in the protease domain, which is normally disulfide bonded to aCys in the pro-domain of the full length MTSP, is replaced with anotheramino acid; the nucleic acid molecule contains a stop codon so that onlythe single chain MTSP protease domain, or proteolytically activefragment thereof, can be expressed; and the MTSP protease domain, orproteolytically active fragment thereof, has serine protease activity asa single chain.
 2. The nucleic acid molecule of claim 1, wherein theMTSP portion has an N-terminus that comprises IVNG, ILGG, VGLL or ILGG.3. The nucleic acid molecule of claim 1, wherein the MTSP is selectedfrom among MTSP1, MTSP3, MTSP4 and MTSP6.
 4. The nucleic acid moleculeof claim 1, wherein the MTSP protease domain consists of a sequence ofamino acid residues selected from among amino acids 615-855 of SEQ IDNO: 2, amino acids 205-437 of SEQ ID NO: 4, the amino acids set forth asSEQ ID NO: 6 or as amino acids 217-443 in SEQ ID NO:
 12. 5. The nucleicacid molecule of claim 1, wherein the encoded polypeptide has at leastabout 95% sequence identity with a protease domain consisting of asequence of amino acid residues selected from among amino acids 615-855of SEQ ID NO: 2, amino acids 205-437 of SEQ ID NO: 4, the amino acidsset forth as SEQ ID NO: 6 and amino acids 217-443 in SEQ ID NO:
 12. 6.The nucleic acid molecule of claim 1, wherein the MTSP protease domainis encoded by a sequence of nucleic acid residues selected from amongnucleotides 1865-2585 of SEQ ID NO: 1, nucleotides 873-1571 of SEQ IDNO: 3, the nucleotides set forth as SEQ ID NO: 5 and nucleotides916-1596 of SEQ ID NO:
 11. 7. The nucleic acid molecule of claim 1,wherein a free Cys in the protease domain of the polypeptide is replacedwith a serine.
 8. The nucleic acid molecule of claim 1, wherein the MTSPis selected from among corin, MTSP1, enteropeptidase, human airwaytrypsin-like protease (HAT), TMPRSS2, and TMPRSS4.
 9. The nucleic acidmolecule of claim 1, wherein: the encoded MTSP protease domain is linkeddirectly or via a polypeptide linker to a targeting agent; and theencoded conjugate has serine protease activity.
 10. The nucleic acidmolecule of claim 1, wherein the nucleic acid encoding the MTSP proteasedomain is operatively linked to a nucleic acid encoding a signalsequence.
 11. The nucleic acid molecule of claim 9, wherein thetargeting agent permits: i) affinity isolation or purification of theconjugate; ii) attachment of the conjugate to a surface; iii) detectionof the conjugate; or iv) targeted delivery to a selected tissue or cell.12. A vector, comprising the nucleic acid molecule of claim
 1. 13. Acell, comprising the vector of claim
 12. 14. The cell of claim 13 thatis a eukaryotic cell.
 15. The cell of claim 13 that is a yeast cell. 16.The cell of claim 15 that is a Pichia species cell.
 17. A method forproduction of a single chain polypeptide that consists only of aprotease domain of a type-II membrane-type serine protease (MTSP), or aproteolytically active fragment thereof, as a single chain, comprising:growing a cell of claim 13 under conditions, whereby the single chainMTSP polypeptide is expressed; and isolating the single chain MTSPpolypeptide.
 18. The method of claim 17, wherein the nucleic acidencoding the MTSP polypeptide is operatively linked to a nucleic acidencoding a signal sequence, whereby the encoded MTSP polypeptide issecreted into the cell culture medium.
 19. The method of claim 17,wherein the cell is a eukaryotic cell.
 20. The method of claim 17,wherein the cell is a yeast cell.
 21. The method of claim 20, whereinthe cell is a Pichia species cell.